Note: Descriptions are shown in the official language in which they were submitted.
CA 02526534 2010-01-11
WO 2005/007081 FCT/US2004/016401
COMPOUNDS AND METHODS FOR DELIVERY OF PROSTACYCLIN
ANALOGS
FIELD OF THE INVENTION
This invention pertains generally to prostacyclin analogs and methods for
their
use in promoting vasodilation, inhibiting platelet aggregation and thrombus
formation, stimulating thrombolysis, inhibiting cell proliferation (including
vascular
remodeling), providing cytoprotection, preventing atherogenesis and inducing
angiogenesis. Through these prostacyclin-mimetic mechanisms, the compounds of
the present invention may be used in the treatment of/for: pulmonary
hypertension,
ischemic diseases (e.g., peripheral vascular disease, Raynaud's phenomenon,
Scleroderma, myocardial ischemia, ischenic stroke, renal insufficiency), heart
failure
(including congestive heart failure), conditions requiring anticoagulation
(e.g., post
MI, post cardiac surgery), thrombotic microangiopathy, extracorporeal
circulation,
central retinal vein occlusion, atherosclerosis, inflammatory diseases (e.g.,
COPD,
psoriasis), hypertension (e.g., preeclampsia), reproduction and parturition,
cancer or
other conditions of unregulated cell growth, cell/tissue preservation and
other
emerging therapeutic areas where prostacyclin treatment appears to have a
beneficial
role. These compounds may also demonstrate additive or synergistic benefit in
1
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
combination with other cardiovascular agents (e.g., calcium channel blockers,
phosphodiesterase inhibitors, endothelial antagonists, antiplatelet agents).
BACKGROUND OF THE INVENTION
Many valuable pharmacologically active compounds cannot be effectively
administered orally for various reasons and are generally administered via
intravenous
or intramuscular routes. These routes of administration generally require
intervention
by a physician or other health care professional, and can entail considerable
discomfort as well as potential local trauma to the patient.
One example of such a compound is treprostinil, a chemically stable analog of
prostacyclin. Although treprostinil sodium (Remodulin ) is approved by the
Food
and Drug Administration (FDA) for subcutaneous administration, treprostinil as
the
free acid has an absolute oral bioavailability of less than 10%. Accordingly,
there is
clinical interest in providing treprostinil orally.
Thus, there is a need for a safe and effective method for increasing the
systemic availability of treprostinil via administration of treprostinil or
treprostinil
analogs.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a compound having structure
I:
O R3
-1n1IIlOR2
H
OCH2CO2R1
2
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
wherein,
R1 is independently selected from the group consisting of H, substituted and
unsubstituted benzyl groups, and groups wherein OR' are substituted or
unsubstituted
glycolamide esters;
R2 and R3 may be the same or different and are independently selected from
the group consisting of H, phosphate and groups wherein OW and OW form esters
of
amino acids or proteins, with the proviso that all of R1, R2 and R3 are not H;
an enantiomer of the compound;
and pharmaceutically acceptable salts of the compound and polymorphs.
In some of these embodiments, R1 is a substituted or unsubstituted benzyl
group, such as CH2C6H5. In other embodiments, OR' is a substituted or
unsubstituted
glycolamide ester, R1 is -CH2CONR4R5, R4 and R5 may be the same or different
and
are independently selected from the group consisting of H, OH, substituted and
unsubstituted alkyl groups, -(CH2),,,CH3, -CH2OH, and -CH2(CH2)õ OH, with the
proviso that in is 0, 1, 2, 3 or 4, and n is 0, 1, 2, 3 or 4. In certain of
these
embodiments one or both of R4 and R5 are independently selected from the group
consisting of H, -OH, -CH3, or -CH2CH2OH. In any of the previously discussed
embodiments, one or both of R2 and R3 can be H. In some enantiomers of the
compound R1=R2=R3=H, or R2=R3=H and R1=valinyl amide.
In still further embodiments of the present compounds R2 and R3 are
independently selected from phosphate and groups wherein OW and OW are esters
of
amino acids, dipeptides, esters of tripeptides and esters of tetrapeptides. In
some
compounds only one of R2 or R3 is a phosphate group. In other compounds R2 and
R3
are independently selected from groups wherein OR2 and OR3 are esters of amino
acids, such as esters of glycine or alanine. In any of the above embodiments,
one of
R2 and R3 are H. In certain of the present compounds, the oral bioavailability
of the
compound is greater than the oral bioavailability of treprostinil, such as at
least 50%
or 100% greater than the oral bioavailability of treprostinil. The above
compounds
can further comprise an inhibitor of p-glycoprotein transport. Any of these
compounds can also further comprise a pharmaceutically acceptable excipient.
3
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
The present invention also provides a method of using the above compounds
therapeutically of/for: pulmonary hypertension, ischemic diseases, heart
failure,
conditions requiring anticoagulation, thrombotic microangiopathy,
extracorporeal
circulation, central retinal vein occlusion, atherosclerosis, inflammatory
diseases,
hypertension, reproduction and parturition, cancer or other conditions of
unregulated
cell growth, cell/tissue preservation and other emerging therapeutic areas
where
prostacyclin treatment appears to have a beneficial role. A preferred
embodiment is a
method of treating pulmonary hypertension and/or peripheral vascular disease
in a
subject comprising orally administering a pharmaceutically effective amount of
a
compound of structure II:
H
O R3
-11,1111OR2
H
OCH2CO2R1
wherein,
R1 is independently selected from the group consisting of H, substituted and
unsubstituted alkyl groups, arylalkyl groups and groups wherein OR1 form a
substituted or unsubstituted glycolamide ester;
R2 and R3 may be the same or different and are independently selected from
the group consisting of H, phosphate and groups wherein OR2 and OR3 form
esters of
amino acids or proteins, with the proviso that all of R1, R2 and R3 are not H;
an enantiomer of the compound; and
a pharmaceutically acceptable salt or polymorph of the compound.
In some of these methods, when OR1 forms a substituted or unsubstituted
glycolamide ester, R1 is -CH2CONR4R5, wherein R4 and R5 may be the same or
different and are independently selected from the group consisting of H, OH,
4
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
substituted and unsubstituted alkyl groups, -(CH2),,,CH3, -CH2OH, and -
CH2(CH2)õ OH, with the proviso that m is 0, 1, 2, 3 or 4, and n is 0, 1, 2, 3
or 4. In
other methods R1 is a C1-C4 alkyl group, such as methyl, ethyl, propyl or
butyl. In the
disclosed methods, R1 can also be a substituted or unsubstituted benzyl group.
In
other methods, R1 can be -CH3 or -CH2C6H5. In still other methods R4 and R5
are the
same or different and are independently selected from the group consisting of
H, OH,
-CH3, and -CH2CH2OH. In yet other methods, one or both of R2 and R3 are H.
Alternatively, one or both of R2 and R3 are not H and R2 and R3 are
independently
selected from phosphate and groups wherein OR2 and OR3 are esters of amino
acids,
dipeptides, esters of tripeptides and esters of tetrapeptides. In some
methods, only
one of R2 or R3 is a phosphate group. In additional methods, R2 and R3 are
independently selected from groups wherein OR2 and OR3 are esters of amino
acids,
such as esters of glycine or alanine. In further methods one of R1 and R2 is
H. In
some methods, enantiomers of the compound where R1=R2=R3=H, or R2=R3=H and
R1=valinyl amide are used.
In various methods the oral bioavailability of the compound is greater than
the
oral bioavailability of treprostinil, such as at least 50% or 100% greater
than the oral
bioavailability of treprostinil. The present methods can also comprise
administering
pharmaceutically effective amount of a p-glycoprotein inhibitor,
simultaneously,
sequentially, or prior to administration of the compound of structure II. In
some
embodiments the p-glycoprotein inhibitor is administered orally or
intravenously.
The disclosed methods can be used to treat pulmonary hypertension.
The present invention also provides a method of increasing the oral
bioavailability of treprostinil or pharmaceutically acceptable salt thereof,
comprising
administering a pharmaceutically effective amount of a p-glycoprotein
inhibitor and
orally administering a pharmaceutically effective amount of treprostinil to a
subject.
In certain of these embodiments the p-glycoprotein inhibitor is administered
prior to
or simultaneously with the treprostinil. The route of the p-glycoprotein
inhibitor
administration can vary, such as orally or intravenously. The present
invention also
provides a composition comprising treprostinil or a pharmaceutically
acceptable salt
thereof and a p-glycoprotein inhibitor.
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
The present compound can also be administered topically or transdermally.
Pharmaceutical formulations according to the present invention are provided
which include any of the compounds described above in combination with a
pharmaceutically acceptable carrier.
The compounds described above can also be used to treat cancer.
Further objects, features and advantages of the invention will be apparent
from
the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B respectively show plasma concentration versus time curves
for intravenous and intraportal dosing of treprostinil diethanolamine salt in
rats as
described in Example 1;
Figures 2A, 2B and 2C respectively show plasma concentration versus time
curves for intraduodenal, intracolonic and oral dosing of treprostinil
diethanol amine
salt in rats as described in Example 1;
Figure 3 shows on a logarithmic scle the average plasma concentration versus
time curves for the routes of administration described in Example 1;
Figure 4 is a graphical representation of the plasma concentration versus time
curve for treprostinil in rat following oral administration in rats of
treprostinil methyl
ester as described in Example 2;
Figure 5 is a graphical representation of the plasma concentration versus time
curve for treprostinil in rat following oral administration in rats of
treprostinil benzyl
ester as described in Example 2;
Figure 6 is a graphical representation of the plasma concentration versus time
curve for treprostinil in rat following oral administration in rats of
treprostinil
diglycine as described in Example 2;
Figure 7 is a graphical representation of the plasma concentration versus time
curve for treprostinil in rat following oral administration in rates of
treprostinil benzyl
6
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
ester (0.5-mg/kg) and treprostinil diglycine (0.5 mg/kg) as described in
Example 2
compared to treprostinil (1 mg/per kg).
Figure 8 is a graphical representation of the plasma concentration versus time
curve for treprostinil in rat following intraduodenal administration of
treprostinil
monophosphate (ring) as described in Example 3;
Figure 9 is a graphical representation of the plasma concentration versus time
curve for treprostinil in rat following intraduodenal administration of
treprostinil
monovaline (ring) as described in Example 3;
Figure 10 is a graphical representation of the plasma concentration versus
time
curve for treprostinil in rat following intraduodenal administration of
treprostinil
monoalanine (ring) as described in Example 3;
Figure 11 is a graphical representation of the plasma concentration versus
time
curve for treprostinil in rat following intraduodenal administration of
treprostinil
monoalanine (chain) as described in Example 3; and
Figure 12 is a graphical representation of the avergage plasma concentration
versus time curve for each prodrug compared to treprostinil alone from Example
1, as
described in Example 3. Treprostinil was dosed at 1 mg/kg whereas the prodrugs
were
dosed at 0.5 mg/kg.
Figures 13A - 13D respectively show doses, administered every two hours for
four doses, for either 0.05 mg per dose (total = 0.2 mg), 0.125 mg per dose
(total = 0.5
mg), 0.25 mg per dose (total = 1.0 mg), or 0.5 mg per dose (total = 2.0 mg).
Figure 14 shows pharmacokinetic profiles of UT-15C sustained release tablets
and sustained release capsules, fasted and fed state.
Figure 15 shows an X ray powder diffraction spectrum of the polymorph Form
A.
Figure 16 shows an IR spectrum of the polymorph Form A.
Figure 17 shows a Raman spectrum of the polymorph Form A.
Figure 18 shows thermal data of the polymorph Form A.
7
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Figure 19 shows moisture sorption data of the polymorph Form A.
Figure 20 shows an X ray powder diffraction spectrum of the polymorph Form
B.
Figure 21 shows thermal data of the polymorph Form B.
Figure 22 shows moisture sorption data of the polymorph Form B.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise specified, "a" or "an" means "one or more". The present
invention provides compounds and methods for inducing prostacyclin-like
effects in a
subject or patient. The compounds provided herein can be formulated into
pharmaceutical formulations and medicaments that are useful in the methods of
the
invention. The invention also provides for the use of the compounds in
preparing
medicaments and pharmaceutical formulations and for use of the compounds in
treating biological conditions related to insufficient prostacyclin activity
as outlined in
the Field of Invention. The present invention also provides compounds and
methods
for the treatment of cancer and cancer related disorders.
In some embodiments, the present compounds are chemical derivatives of (+)-
treprostinil, which has the following structure:
OH
-1111110H
qTH
OCH2CO2H
Treprostinil is a chemically stable analog of prostacyclin, and as such is a
potent vasodilator and inhibitor of platelet aggregation. The sodium salt of
treprostinil, (1R,2R,3aS,9aS)-[[2,3,3a,4,9,9a -Hexahydro-2-hydroxy -1-[(3S)-3-
hydroxyoctyl]-1H-benz[f]inden-5-yl] oxy]acetic acid monosodium salt, is sold
as a
8
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
solution for injection as Remodulin which has been approved by the Food and
Drug
Administration (FDA) for treatment of pulmonary hypertension. In some
embodiments, the present compounds are derivatives of (-)-treprostinil, the
enantiomer of (+)-treprostinil. A preferred embodiment of the present
invention is the
diethanolamine salt of treprostinil. The present invention further includes
polymorphs
of the above compounds, with two forms, A and B, being described in the
examples
below. Of the two forms, B is preferred. A particularly preferred embodiment
of the
present invention is form B of treprostinil diethanolamine.
In some embodiments, the present compounds are generally classified as
prodrugs of treprostinil that convert to treprostinil after administration to
a patient,
such as through ingestion. In some embodiments, the prodrugs have little or no
activity themselves and only show activity after being converted to
treprostinil. In
some embodiments, the present compounds were produced by chemically
derivatizing
treprostinil to make stable esters, and in some instances, the compounds were
derivatized from the hydroxyl groups. Compounds of the present invention can
also
be provided by modifying the compounds found in U.S. Patent Nos. 4,306,075 and
5,153,222 in like manner.
In one embodiment, the present invention provides compounds of structure I:
H
OR3
-11111OR2
H
OCH2CO2R1
wherein,
R1 is independently selected from the group consisting of H, substituted and
unsubstituted benzyl groups and groups wherein OR' are substituted or
unsubstituted
glycolamide esters;
9
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
R2 and R3 may be the same or different and are independently selected from
the group consisting of H, phosphate and groups wherein OR2 and OR3 form
esters of
amino acids or proteins, with the proviso that all of R1, R2 and R3 are not H;
enantiomers of the compound; and
pharmaceutically acceptable salts of the compound.
In some embodiments wherein OR' are substituted or unsubstituted
glycolamide esters, R1 is -CH2CONR4R5 and R4 and R5 may be the same or
different
and are independently selected from the group consisting of H, OH, substituted
and
unsubstituted alkyl groups, -(CH2)mCH3, -CH2OH, and -CH2(CH2)õ OH, with the
proviso that in is 0, 1, 2, 3 or 4, and n is 0, 1, 2, 3 or 4.
One skilled in the art will also readily recognize that where members are
grouped together in a common manner, such as in a Markush group or the groups
described in the R of structures I and II above and below,'the present
invention
encompasses not only the entire group listed as a whole, but each member of
the
group individually and all possible subgroups of the main group. Accordingly,
for all
purposes, the present invention encompasses not only the main group, but also
the
main group absent one or more of the group members. The present invention also
envisages the explicit exclusion of one or more of any of the group members in
the
claimed invention. For example, R1 can specifically exclude H, substituted and
unsubstituted benzyl groups, or groups wherein OR1 are substituted, or
unsubstituted
glycolamide esters.
In some embodiments, R1 is a substituted or unsubstituted benzyl groups, such
as -CH2C6H5, -CH2C6H4NO2, -CH2C6H4OCH3, -CH2C6H4C1, -CH2C6H4(NO2)2, or -
CH2C6H4F. The benzyl group can be ortho, meta, para, ortho/para substituted
and
combinations thereof. Suitable substituents on the aromatic ring include
halogens
(fluorine, chlorine, bromine, iodine), -NO2 -OR16 groups wherein R16 is H or
a C,-C4 alkyl group, and combinations thereof.
Alternatively, when R' is -CH2CONR4R5 then R4 and R5 may be the same or
different
and are independently selected from the group consisting of H, OH, -CH3, and -
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
CH2CH2OH. In these compounds where R1 is not H, generally one or both of R2
and
R3 are H. t
In some embodiment one or both of R2 and R3 are H and R1 is -CH2CONR4R5,
and one or both of R4 and R5 are H, -OH, -CH3, -CH2CH2OH.
In compounds where one or both of R2 and R3 are not H, R2 and R3 can be
independently selected from phosphate and groups wherein OR2 and OR3 are
esters of
amino acids, dipeptides, esters of tripeptides and esters of tetrapeptides. In
some
embodiments, only one of R2 or R3 is a phosphate group. In compounds where at
least one of R2 and R3 is not H, generally R1 is H. In additional embodiments,
one of
R2 and R3 are H and thus the compound of structure I is derivatized at only
one of R2
and W. In particular compounds, R2 is H and R3 is defined as above. In
additional
embodiments, R1 and R3 are H and R2 is a group wherein OR2 is an ester of an
amino
acid or a dipeptide. In further embodiments, R1 and R2 are H and R3 is a group
wherein OR3 is an ester of an amino acid or a dipeptide.
When one or both of the OR2 and OR3 groups form esters of amino acids or
peptides, i.e., dipeptides, tripeptides or tetrapeptides, these can be
depicted generically
as -COCHR6NR7R8 wherein R6 is selected from the group consisting of amino acid
side chains, R7 and R8 may be the same or different and are independently
selected
from the group consisting of H, and -COCHR9NR10R11. Generally, reference to
amino acids or peptides refers to the naturally occurring, or L-isomer, of the
amino
acids or peptides. However, the present compounds and methods are not limited
thereto and D-isomer amino acid residues can take the place of some or all of
L-
amino acids. In like manner, mixtures of D- and L-isomers can also be used. In
the
embodiments wherein the amino acid is proline, R7 together with R6 forms a
pyrrolidine ring structure. R6 can be any of the naturally occurring amino
acid side
chains, for example -CH3 (alanine), -(CH2)3NHCNH2NH (arginine), -CH2CONH2
(asparagine), -CH2COOH (aspartic acid,), -CH2SH (cysteine), -(CH2)2CONH2
(glutamine), -(CH2)2COOH (glutamic acid), -H (glycine), -CHCH3CH2CH3
(isoleucine), -CH2CH(CH3)2 (leucine), -(CH2)4NH2 (lysine), -(CH2)2SCH3
(methionine), -CH2Ph (phenylalanine), -CH2OH (serine), -CHOHCH3 (threonine), -
CH(CH3)2 (valine),
11
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
H
N
H NH
(histidine),
(tryptophan), and
N ~~j
H
2
H2
OH
(t1'r'osine),
-(CH2)3NHCONH2 (citrulline) or -(CH2)3NH2 (ornithine). Ph designates a phenyl
group.
In the above compounds, Wand R8 may be the same or different and are
selected from the group consisting of H, and -COCHR9NR10R11, wherein R9 is a
side
chain of amino acid, R10 and R11 maybe the same or different and are selected
from
the group consisting of H, and -COCHR12NR13R14, wherein R12 is an amino acid
side
chain, R13 and R14 may be the same or different and are independently selected
from
the group consisting of H, and -COCHR15NH2. One skilled in the art will
realize that
the peptide chains can be extended on the following scheme to the desired
length and
include the desired amino acid residues.
In the embodiments where either or both of OR2 and OR3 groups form an ester
of a peptide, such as dipeptide, tripeptide, tetrapeptide, etc, the peptides
can be either
homopeptides, i.e., repeats of the same amino acid, such as arginyl-arginine,
or
heteropeptides, i.e., made up of different combinations of amino acids.
Examples of
heterodipeptides include alanyl-glutamine, glycyl-glutamine, lysyl-arginine,
etc.
As will be understood by the skilled artisan when only one R7 and R8 includes
a peptide bond to further amino acid, such as in the di, tri and
tetrapeptides, the
resulting peptide chain will be linear. When both R7 and R8 include a peptide
bond,
then the peptide can be branched.
12
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
In still other embodiments of the present compounds Rl is H and one of R2 or
R3 is a phosphate group or H while the other R2 or R3 is a group such the OR2
or OR3
is an ester of an amino acid, such as an ester of glycine or alanine.
Pharmaceutically acceptable salts of these compounds as well as
pharmaceutical formulation of these compounds are also provided.
Generally, the compounds described herein have enhanced oral bioavailability
compared to the oral bioavailability of treprostinil, either in free acid or
salt form.
The described compounds can have oral bioavailability that is at least 25%,
50%
100%, 200%, 400% or more compared to the oral bioavailability of treprostinil.
The
absolute oral bioavailability of these compounds can range between 10%, 15%,
20%,
25%, 30% and 40%,45%,50%,55%,60% or more when administered orally. For
comparison, the absolute oral bioavailability of treprostinil is on the order
of 10%,
although treprostinil sodium has an absolute bioavailability approximating
100%
when administered by subcutaneous infusion.
As will be understood by one skilled in the art, for any and all purposes,
particularly in terms of providing a written description, all ranges disclosed
herein,
and in particular the bioavailability ranges described herein also encompass
any and
all possible subranges and combinations of subranges thereof. As only one
example,
a range of 20% to 40%, can be broken down into ranges of 20% to 32.5% and
32.5%
to 40%, 20% to 27.5% and 27.5% to 40%, etc. Any listed range can be easily
recognized as sufficiently describing and enabling the same range being broken
down
into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-
limiting
example, each range discussed herein can be readily broken down into a lower
third,
middle third and upper third, etc. As will also be understood by one skilled
in the art
all language such as "up to," "at least," "greater than," "less than," "more
than" and
the like include the number recited and refer to ranges which can be
subsequently
broken down into subranges as discussed above. In the same manner, all ratios
disclosed herein also include all subratios falling within the broader ratio.
Administration of these compounds can be by any route by which the
compound will be bioavailable in effective amounts including oral and
parenteral
13
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
routes. The compounds can be administered intravenously, topically,
subcutaneously,
intranasally, rectally, intramuscularly, transdermally or by other parenteral
routes.
When administered orally, the compounds can be administered in any convenient
dosage form including, for example, capsule, tablet, liquid, suspension, and
the like.
Testing has shown that that treprostinil can be irritating upon skin contact.
In
contrast, some of the compounds disclosed herein, generally as prodrugs of
treprostinil, are not irritating to the skin. Accordingly, the present
compounds are
well suited for topical or transdermal administration.
When administered to a subject, the above compounds, and in particular the
compounds of structure I, are prostacyclin-mimetic and are useful in treating
conditions or disorders where vasodilation and/or inhibition of platelet
aggregation or
other disorders where prostacyclin has shown benefit, such as in treating
pulmonary
hypertension. Accordingly, the present invention provides methods for inducing
prostacyclin-like effects in a subject comprising administering a
pharmaceutically
effective amount of one or more of the compounds described herein, such as
those of
structure I above, preferably orally, to a patient in need of such treatment.
As an
example, the vasodilating effects of the present compounds can be used to
treat
pulmonary hypertension, which result from various forms of connective tissue
disease, such as lupus, sclerodenna or mixed connective tissue disease. These
compounds are thus useful for the treatment of pulmonary hypertension.
In another embodiment, the present invention also provides methods of
promoting prostacyclin-like effect in a subject by administering a
pharmaceutically
effective amount of a compound of structure II:
H
OR3
H
OCH2CO2R1
14
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
wherein,
R1 is independently selected from the group consisting of H, substituted and
unsubstituted alkyl groups, arylalkyl groups and groups wherein OR1 form a
substituted or unsubstituted glycolamide ester;
R2 and R3 may be the same or different and are independently selected from
the group consisting of H, phosphate and groups wherein OR2 and OR3 form
esters of
amino acids or proteins, with the proviso that all of R1, R2 and R3 are not H;
an enantiomer of the compound; and
a pharmaceutically acceptable salt of the compound.
In groups wherein OR' form a substituted or unsubstituted glycolamide ester,
RI can be -CH2CONR4R5, wherein R4 and R5 may be the same or different and are
independently selected from the group consisting of H, OH, substituted and
unsubstituted alkyl groups, -(CH2)mCH3, -CH2OH, and -CH2(CH2)õOH, with the
proviso that m is 0, 1, 2, 3 or 4, and n is 0, 1, 2, 3 or 4.
In other methods of inducing vasodilation or treating hypertension, Ri can be
a
C1-C4 alkyl group, such as methyl, ethyl, propyl or butyl. In other methods R1
is a
substituted or unsubstituted benzyl groups, such as -CH2C6H5, -CH2C6H4NO2, -
CH2C6H4OCH3, -CH2C6H4C1, -CH2C6H4(NO2)2, or -CH2C6H4F. The benzyl group
can be ortho, meta, para, ortho/para substituted and combinations thereof.
Suitable
substituents on the aromatic ring include halogens (fluorine, chlorine,
bromine,
iodine), -NO2 -OR16 groups wherein R16 is H or a C1-C4 alkyl group, and
combinations thereof.
Alternatively, when R1 is -CH2CONR4R5 then R4 and R5 may be the same or
different and are independently selected from the group consisting of H, OH, -
CH3,
and -CH2CH2OH. In these methods, where R1 is not H, generally one or both of
R2
and R3 are H.
In some methods, one or both of R2 and R3 are H and R1 is -CH3, -CH2C6H5.
In other methods where one or both of R2 and R3 are H, then R1 is -CH2CONR4R5,
and one or both of R4 and R5 are H, -OH, -CH3, -CH2CH2OH.
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
In methods where one or both of R2 and R3 are not H, R2 and R3 can be
independently selected from phosphate and groups wherein OR2 and OR3 are
esters of
amino acids, dipeptides, esters of tripeptides and esters of tetrapeptides. In
some
embodiments, only one of R2 or R3 is a phosphate group. In methods where at
least
one of R2 and R3 is not H, generally R1 is H. In other methods, one of R2 or
R3 is H
and the other R2 or R3 is as defined elsewhere herein. In some methods, R2 is
H and
R3 is not H. In additional embodiments, R' and R3 are H and R2 is a group
wherein
OR2 is an ester of an amino acid or a dipeptide. In further embodiments, R1
and R2
are H and R3 is a group wherein OR3 is an ester of an amino acid or a
dipeptide.
In the methods, where one or both of the OR2 and OR3 groups form esters of
amino acids or peptides, i.e., dipeptides, tripeptides or tetrapeptides, these
can be
depicted generically as -COCHR6NR7R8 wherein R6 is selected from the group
consisting of amino acid side chains, R7 and R8 may be the same or different
and are
independently selected from the group consisting of H, and -COCHR9NR10R11 In
the
embodiments wherein the amino acid is proline, R7 together with R6 forms a
pyrrolidine ring structure. R6 can be any of the naturally occurring amino
acid side
chains, for example -CH3 (alanine), -(CH2)3NHCNH2NH (arginine), -CH2CONH2
(asparagine), -CH2COOH (aspartic acid,), -CH2SH (cysteine), -(CH2)2CONH2
(glutamine), -(CH2)2COOH (glutamic acid), -H (glycine), -CHCH3CH2CH3
(isoleucine), -CH2CH(CH3)2 (leucine), -(CH2)4NH2 (lysine), -(CH2)2SCH3
(methionine), -CH2Ph (phenylalanine), -CH2OH (serine), -CHOHCH3 (threonine), -
CH(CH3)2 (valine),
H
N
NH
H2
C (histidine),
(tryptophan), and
H2
C \ OH sine
16
CA 02526534 2010-01-11
WO 2005/007081 PCT/US2004/016401
-(CH2)3NHCONH2 (citrulline) or -(CH2)3NH2 (ornithine). Ph designates a phenyl
group.
In the above methods, R7 and R8 may be the same or different and are selected
from the group consisting of H, and -COCHR9NR10R11, wherein R9 is a side chain
of
amino acid, R10 and R11 may be the same or different and are selected from the
group
consisting of H, and -COCHR17NR13R14, wherein R12 is an amino acid side chain,
R13
and R14 may be the same or different and are independently selected from the
group
consisting of H, and -COCHR15NH2. One skilled in the art will realize that the
peptide chains can be extended on the following scheme to the desired length
and
include the desired amino acid residues.
In the embodiments where either or both of OR2 and OR3 groups form an ester
of a peptide, such as dipeptide, tripeptide, tetrapeptide, etc. the peptides
can be either
homopeptides, i.e., repeats of the same amino residue, or heteropeptides,
i.e., made up
of different combinations of amino acids.
As will be understood by the skilled artisan when only one of R7 and R8
includes a peptide bond to further amino acid, such as in the di, tri and
tetrapeptides,
the resulting peptide chain will be linear. When both R7 and R8 include a
peptide
bond, then the peptide can be branched.
In still other methods R1 is H and one of R2 or R3 is a phosphate group or H
while the other R2 or R3 is a group such the OR2 or OR3 is an ester of an
amino acid,
such as an ester of glycine or alanine.
In some methods, the administered compound can have an oral bioavailability
that is at least 25%, 50% 100%, 200%, 400% of the oral bioavailability of
treprostinil.
It is generally preferred to administer compounds that have higher absolute
oral
bioavailabilities, such as 15%, 20%, 25%, 30% and 40%, 45%, 50%, 55%,'60% or
more when administered orally.
Treprostinil has also been discovered to inhibit metastasis of cancer cells
Accordingly, the compounds described above, and
17
CA 02526534 2010-01-11
WO 2005/007081 PCT/US2004/016401
in particular those of structure I and II, can also be used in the treatment
of cancer and
cancer related disorders, and as such the present invention provides
pharmaceutical
compositions and methods for treating cancer. Suitable formulations and
methods of
using the present compounds can be achieved by substituting the compounds of
the
present invention, such as those of structure I and II and in particular
prodrugs of
treprostinil, for the active compounds
Synthesis of the following compounds of structure I and structure II can be
achieved as follows:
Synthesis of methyl ester of Treprostinil (2) and biphosphate ester of
Treprostinil
H
H e H off (NCCH2CH2O)P(O)(OH)3 OR
off DCC, Py _ .......OH
mOH
=^^OH CIi,OH
HG
O H
O
H ` I
O 1
COOCII3 COOCH,
3 R - R'- P(O)(0H)(OCHCHCN)
2
COOI{
1 I) NaOH/Me0-H2o
2) Dowoz H'
H
6R
(O)OR'
O
000H
4Rw R'..P(O)(OH),
Synthesis of methyl ester of Treprostinil (2)
Methyl ester of treprostinil (2) was prepared by treating 1.087 g (2.8 nu-
holes)
of treprostinil (1) with 50 ml of a saturated solution of dry hydrochloric
acid in
methanol. After 24 hours at room temperature, the methanol was evaporated to
dryness and the residue was taken in 200 ml dichloromethane. The
dichloromethane
solution was washed with a 10% aqueous potassium carbonate solution, and then
with
18
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
water to a neutral pH, it was dried over sodium sulfate, filtered and the
solvent was
removed in vacuo affording treprostinil methyl ester (2) in 98% yield as a
yellow oil.
The crude methyl ester was used as such in subsequent reactions.
Synthesis of biphosphate ester of Treprostinil (4)
The procedure was adapted after Steroids, 2(6), 567-603(1963). The methyl
ester of treprostinil (2) (60 mg, 0.15 minoles) was dissolved in 2 ml dry
pyridine and a
pyridinium solution of the previously prepared pyridinium solution of 2-
cyanoethylphosphate 1M (0.3 ml, 0.3 irumoles) (cf. Methods in Enzymology,
1971,
18(c), 54-57) were concentrated to dryness in vacuo at 40 C. Anhydrous
pyridine
was added and the reaction mixture was again concentrated; the operation was
repeated twice in order to remove water completely. Finally the residue was
dissolved in 2 ml anhydrous pyridine and 190 mg (0.9 mmoles)
dicyclohexylcarbodiimide were added as a solution in 2 ml anhydrous pyridine.
The
reaction mixture in a closed flask was stirred magnetically for 48 hours at
room
temperature. lml water was added and after one hour, the mixture was
concentrated
to a thick paste in vacuo. The reaction mixture was treated overnight at room
temperature with 3 ml of a 1/9 water/methanol solution containing 35 mg sodium
hydroxide. The white solid (dicyclohexylurea) formed was removed by filtration
and
it was washed well with water. The aqueous-methanolic solution was
concentrated
almost to dryness in vacuo, water was added and the solution was extracted
with n-
butanol (3 x 2 ml), then with methylene chloride (1 x 2 ml). The pH of the
solution
was adjusted to 9.0 by treatment with a sulfonic acid ion exchange resin (H+
cycle -
Dowex), treatment with Dowex resin for a longer time (-j 12 hours) lead to
both the
cleavage of the TBDMS group and the recovery of the free carboxyl group. The
resin
was filtered and the solution was concentrated to dryness affording the
corresponding
bisphosphate 4 (43 mg, yield 52%).
19
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Synthesis of 3'-monophosphate ester of treprostinil (8) and 2- monophosphate
ester of treprostinil (10)
OH [BI~MSCt low --Or
eH2Cl~ li HCC, h
O0CIH3
7~COOCH, Y ry ~y CG(X-'H3
/v Y / 9 1~ L
2 6R=H; R' '"112DM 1R= 1p 1.(0)(0H)(OCHHCH2C1); R'= ~Cyn 3UDMS
SR = TBDMS; R' = H 9 R= TBDMS, R' - P(0)(OH)(OCR CH2C N)
U NZORMCOFI Ik0
0 2)Fkz~scaH~
H OIT
H
O'~
COOFF
8R=P(O)(OH)2;R"=H.
1o R =H; R' = P(0)(04
Synthesis of monoprotected TBDMS methyl ester of treprostinil (5 and 6)
The procedure was adapted from Org. Synth., 1998, 75, 139-145. The
treprostinil methyl ester (2) (305.8 mg, 0.75 mmoles) was dissolved in 15 ml
anhydrous dichloromethane and the solution was cooled on an ice bath to 0 C.
Imidazole (102 mg, 1.5 mmoles) and tert-butyldimethyl silyl chloride (226.2
mg, 1.5
mmoles) were added and the mixture was maintained under stirring at 0 C for 30
minutes, then stirred overnight at room temperature. Water (25 ml) was added
and
the organic layer was separated. The aqueous layer was then extracted with
dichloromethane (3 x 50 ml). The organic layers were dried over Na2SO4, the
solution
was filtered and the solvent was removed in vacuo affording 447 mg crude
reaction
product. The crude reaction product was separated by column chromatography
(silica
gel, 35% ethyl acetate/hexanes) affording 140 mg bis-TBDMS protected
Treprostinil
methyl ester, 160 mg 2-TBDMS protected treprostinil methyl ester (6) and 60 mg
3'-
TBDMS protected Treprostinil methyl ester (5).
20 j
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Synthesis of monophosphate ester of Treprostinil 8/10
The procedure was adapted after Steroids, 1963, 2(6), 567-603 and is the same
for (8) and (10) starting from (6) and (5), respectively. The TBDMS protected
methyl
ester of treprostinil (6) (46 mg, 0.09 mmoles) was dissolved in 2 ml dry
pyridine and a
pyridinium solution of the previously prepared pyridinium solution of 2-
cyanoethyiphosphate 1M (0.2 ml, 0.2 mmoles) (cf. Methods in Enzymology, 1971,
18(c), 54-57) were concentrated to dryness in vacuo at 40 C. Anhydrous
pyridine
was added and the reaction mixture was again concentrated; the operation was
repeated twice in order to remove water completely. Finally the residue was
dissolved in 2 ml anhydrous pyridine and 116 mg (0.56 mmoles)
dicyclohexylcarbodiimide were added as a solution in 2 ml anhydrous pyridine.
The
reaction mixture in a closed flask was stirred magnetically for 48 hours at
room
temperature in the dark. 5 ml water were added and after one hour, the mixture
was
concentrated to a thick paste in vacuo. The reaction mixture was treated
overnight at
room temperature with 10 ml of a 1/9 water/methanol solution containing 100 mg
sodium hydroxide. The white solid (dicyclohexylurea) formed was removed by
filtration and it was washed well with water. The aqueous-methanolic solution
was
concentrated almost to dryness in vacuo, water was added and the solution was
extracted with n-butanol (3 x 10 ml), then with methylene chloride (1 x 10
ml). The
pH of the solution was adjusted to 9.0 by treatment with a sulfonic acid ion
exchange
resin (H+ cycle - Dowex); treatment with Dowex resin for a longer time (-- 12
hours)
lead to both the cleavage of the TBDMS group and the recovery of the free
carboxyl
group. The resin was filtered and the solution was concentrated to dryness
affording
the corresponding monophosphate 8 (33 mg, yield 68%).
21
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Synthesis of methyl ester of treprostinil (2)
D0H
H
OCH2COOCH3
2
(2) (1 g; 2.56 mmol) was added to methanol (50 ml) prior saturated with
gaseous hydrochloric acid and the mixture swirled to give a clear solution
that was
left to stand overnight at room temperature. Solvent was removed in vacuo and
the
residue was neutralized with a 20% potassium carbonate solution and extracted
in
dichloroinethane. The organic layer was washed with water, dried over
anhydrous
magnesium sulfate and evaporated to yield the crude product (0.96 g).
Purification by
preparative tlc (silica gel plate; eluent: 7:3 (v/v) hexane-ethyl acetate)
afforded 2
(0.803; 77.5%), colorless oil.
Synthesis of Tritreprostinil diethanolamine (UT-15C)
Treprostinil acid acid is dissolved in a 1:1 molar ratio mixture of
ethanol:water and
diethanolamine is added and dissolved. The solution is heated and acetone is
added
as an antisolvent during cooling.
22
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Synthesis of diglycil ester of treprostinil methyl ester (12)
ct~ca c~co.acci.cj NO, mow, flcx ex~.NizccZ+
~j OH ~~ }-t~C7C,CHp~I Ct i?CI3, FIs
DMAP
OCH2C00CHj oCIi 00CFiy
2 11
......._, -000.0 ~2.NHZ
12
To a magnetically stirred solution of (2) methyl ester 2 (0.268 g; 0.66 mmol)
in dichloromethane (30 ml) N-carbobenzyloxyglycine p-nitrophenyl ester (0.766
g;
2.32 mmol) and 4-(dimethyamino)pyridine (250 mg; 2.05 mmol) were successively
added. The resulted yellow solution was stirred at 20 C for 24 hrs., then
treated with
5% sodium hydroxide solution (20 ml) and stirring continued for 15 mm.
Dichloromethane (50 ml) was added, layers separated and the organic phase
washed
with a 5% sodium hydroxide solution (6 x 20 ml), water (30 ml), 10%
hydrochloric
acid (2 x 40 ml), 5% sodium bicarbonate solution (40 ml) and dried over
anhydrous
sodium sulfate. Removal of the solvent afforded crude (11) (0.61 g), pale-
yellow
viscous oil. Purification by flash column chromatography on silica gel eluting
with
gradient 9/1 to 1/2 (v/v) hexane-ethyl ether afforded 0.445 g (85.3%) of 11,
white
crystals, m.p. 70-72 C. 'Fl-NMR [CDC13i8(ppm)]: 3.786 (s)(3H, COOCH3), 3.875
(d)(2H) and 3.940 (d)(2H)(NH-CH2-COO), 4.631 (s) (2 H, OCH2CO00H3), 4.789
(m)(1H, adjacent to OOC-CH2NHcbz) and 4.903 (m) (1H, adjacent to
QOCCH2NHcbz), 5.09 (s)(4H, C6H5CH2O), 5.378 (m)(1H) and 5.392 (m)(1H)(NH),
7.295-7.329 (m)(1OH, C6H5). LR ESI-MS (m/z): 787.1 [M+H]+, 804.1 [M+NH4]+,
809.3 [M+Na]+, 825.2 [M+K]+, 1590.5 [2M+NH4]+, 1595.6 [2M+Na]+.
23
CA 02526534 2010-01-11
WO 2005/007081 PCT/US2004/016401
Methyl ester, diglycyl ester (12)
A solution of ester (11) (0.4 g; 0.51 mmol) in methanol (30 ml) was
introduced in the pressure bottle of a Parr hydrogenation apparatus, 10%
palladium on
charcoal (0.2 g; 0.197 mmol Pd) was added, apparatus closed, purged thrice
with
hydrogen and loaded with hydrogen at 50 p.s.i. Stirring was started and
hydrogenation carried out for 5 hrs. at room temperature. Hydrogen aas removed
from the installation by vacuum suction and replaced with argon. The catalyst
was
filtered off through celite deposited on a fit and the filtrate concentrated
in vacuo to
give 0.240 g (91%) of 4, white solid m.p. 98-100 C.
Synthesis of benzyl ester of treprostinil (13)
H
OH
-11111IOH
H
OCH2COOCH2C6H5
13
To a stirred solution of (2) (2 g; 5.12 mmol) in anhydrous tetrahydrofuran {20
ml) benzyl bromide (0.95 ml; 7.98 minol) and freshly distilled triethylamine
(1.6 ml;
11.48 mmol) were consecutively added at room temperature and the obtained
solution
was refluxed with stirring for 12 hrs. A white precipitate was gradually
formed.
Solvent was distilled off in vacuo and the residue treated with water (30 ml).
Upon
extraction with methylene chloride emulsion formation occurs. The organic and
aqueous layers could be separated only after treatment with 5% hydrochloric
acid
solution (20 ml). The organic layer was washed with water, 'dried on anhydrous
sodium sulfate, and evaporated, the residue was further dried under reduced
pressure
over phosphorus pentoxide to give a yellow viscous oil (2.32 g) that was
purified by
* Trade-mark
24
CA 02526534 2010-01-11
WO 2005/007081 PCT/US2004/016401
preparative thin layer chromatography (silica gel plate; eluent: 1:2, v/v,
hexane/ethyl
ether). Yield: 81.2%.
Synthesis of bis-glycyl ester of treprostinil (15)
go:: OH C6H5CH2OC0=NHCH2=COO \ / N02 I \ D:: OOC-CH2=NH=000CH2C6H5
OH OOC=CH2=NH-COOCH2C6H5
DMAP
OCH2000CH2C6H5 OCH2COOCH2C6H5
13
14
000=C-NH2
H2 Pd ql:~SOOC'C"H2'M2
OCH2OOOH
Benzy ester, di-cbzGly ester (14)
To a magnetically stirred solution of benzyl ester 13 (1 g; 2.08 mmol) in
dichloromethane (SO ml) N-carbobenzyloxyglycine p-nitrophenyl ester (2.41 g;
7.28
mmol) and 4-(dimethyamino) pyridine (788 mg; 6.45 mmol) were added. The
resulted yellow solution was stirred at 20 C for 21 hrs., then successively
washed
with a 5% sodium hydroxide solution (6 x 45 ml), 10% hydrochloric acid (2 x 40
ml),
5% sodium bicarbonate solution (40 ml) and dried over anhydrous sodium
sulfate.
Removal of the solvent, followed by drying over phosphorus pentoxide under
reduced
pressure, afforded crude 14 (2.61 g), pale-yellow oil. Purification by flash
column
chromatography on silica gel eluting with gradient 9:1 to 1:2 (v/v) hexane-
ethyl ether
gave (14_ (1.51 g; 84.1%) as a colorless, very viscous oil.
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Diglycyl ester (15)
A solution of ester (14) (0.4 g; 0.46 mmol) in methanol (30 ml) was
hydrogenated over 10% Pd/C as described for ester (12). Work-up and drying
over
phosphorus pentoxide in vacuo yielded 0.170 g (72.7%) of ester 15, white solid
m.p.
155-158 C.
Synthesis of 3'-glycyl ester of treprostinil 19 fin i-a-C(CH3} / + oil
t7CHaCOOCH,C6H5 N,, OCHCOOCHHC6tk
O
1
ii
13 16
~:~II I3 ~?`{?,N}Lc , ;G s~ v 0101.`,t4. =LC`OOC.H2C6I 5
' I
MW
OCH2CO0CH2C6H3
17
~:.Q _ ` . l (l[3 .Ctt NII.CC~OU Crkts ry>~ C 7C G CH Nti
11CI
~~tII I~
Pat
Cll CQOCH;I J[3~ oonycoott
19
Benzyl ester, t-butyldimethysilyl monoester (16)
A solution of tert-butyldimethylsilyl chloride (0.45 g; 2.98 mmol) in
dichloromethane (8 ml) was added dropwise over 10 min., at room temperature,
into a
stirred solution of benzyl ester 13 (0.83 g; 1.73mmol) and imidazole (0.33 g;
4.85
26
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
mmol) in dichloromethane (20 ml). Stirring was continued overnight then water
(20
ml) was added, the mixture stirred for one hour, layers separated, organic
layer dried
over anhydrous sodium sulfate and concentrated in vacuo to give a slightly
yellow oil
(1.15 g). The crude product is a mixture of the mono-TBDMS (16) and di-TBDMS
esters ('H-NMR). Column chromatography on silica gel, eluting with a 9:1 (v/v)
hexane-ethyl acetate mixture, readily afforded the di-ester (0.618 g) in a
first fraction,
and ester 16 (0.353 g; yield relative to 13: 34.4%) in subsequent fractions.
Analytical
tlc on silica gel of the ester 16 showed only one spot (eluent: 3:2 (v/v)
hexane-ethyl
ether). Consequently, under the above reaction conditions, the other possible
isomer
(mono-TBDMS ester at the side-chain hydroxyl) was not observed.
Another experiment in which the molar ratio tert-butyldimethylsilyl chloride:
ester 13 was lowered to 1.49 (followed by flash column chromatography of the
product on silica gel, eluting with gradient 9.5/0.5 to 3/1 (v/v) hexane-ethyl
ether)
lead to a decreased content (36.5%, as pure isolated material) of the
undesired di-
OTBDMS by-product. The mono-OTBDMS ester fractions (45.1 %; isolated
material) consisted of ester 16 (98%) and its side-chain isomer (2%) that
could be
distinctly separated; the latter was evidenced (tlc, NMR) only in the last of
the
monoester fractions.
Benzyl ester, cbz-glycyl monoester (18)
To a magnetically stirred solution of ester 16 (0.340 g; 0.57 mmol) in
dichloromethane (15 ml) N-carbobenzyloxyglycine p-nitrophenyl ester (0.445 g;
1.35
mmol) and 4-(dimethyamino) pyridine (150 mg; 1.23 mmol) were successively
added.
The solution was stirred at 20 C for 40 hrs. Work-up as described for esters
11 and
14 yielded a crude product (0.63 g) containing 90% 17 and 10% 18 (1H-NMR). To
completely remove the protective TBDMS group, this mixture was dissolved in
ethanol (30 ml) and subjected to acid hydrolysis (5% HC1, 7 ml) by stirring
overnight
at room temperature. Solvent was then removed under reduced pressure and the
residue extracted in dichloromethane (3 x 50 ml); the organic layer was
separated,
washed once with water (50 ml), dried over sodium sulfate and concentrated in
vacuo
27
CA 02526534 2010-01-11
WO 2005/007081 PCT/US2004/016401
to give crude ester 18 (0.51 g). Purification by flash column chromatography
as for
esters 11 and 14 afforded ester 18 (0.150 g; overall yield: 39.1%) as a
colorless,
viscous oil.
Glycyl monoester (19)
A solution of ester 18 (0.15 g; 0.22 mmol) in methanol (30 ml) was
hydrogenated over 10% Pd/C as described for ester 12 and 15. Work-up and
drying
over phosphorus pentoxide in vacuo yielded ester 10 (0.98 g; 98.0%), white,
shiny
crystals m.p. 74-76 C. LR ESI-MS (m/z): 448.2 [M+Hj+, 446.4 [M-H]".
Synthesis of 3'-L-leucyl ester of treprostinil 22
0
N-OOC=CHCH2CH(CHz)2
NH=COOCH2C6H5 CH(NHCbz)CH2CH(CII2)2
OH qog')TBC
MS
OT BDMS DMAP
OCH2000CFI2C6H5
OCH2OOOCH2C6H5
,
16
OOC=CH(NHcbz)CH2CH(CH3)2 H2/Pd OOC=CH(NH2)CH3CH(C143)2
1I I OH
IICI _ PC': 0
EtOH, CHC12 OCH1COOCH2C6H5 OCH2OO0H
21
22
Benzyl ester, t-butyldimethysilyl monoester, cbz-L-leucyl monoester (20)
To a stirred solution of ester 16 (0.38 g: 0.64 mmol) and N-carbobenzyloxy-L-
leucine N-hydroxysuccinimide ester (0.37 g; 1.02 mmol) in 10 ml
dichloromethane 4-
(dimethyamino)pyridine (0.17 g; 1.39 mmol) was added, then stirring continued
at
room temperature for 2 days. The solvent was removed in vacuo and the crude
product (0.9 g) subjected to flash column chromatography on silica gel eluting
with
28
CA 02526534 2010-01-11
WO 2005/007081 PCT/US2004/016401
9:1 hexane-ethyl acetate; the firstly collected fraction yielded an oil (0.51
g) which,
based on the its NMR spectrum and tic, was proved to be a 2:1 mixture of ester
20
and the starting ester 16, Preparative tlc on silica gel (eluent: ethyl
acetate-hexane
1:4) gave pure 20, colorless oil (overall yield based on 7: 62.6%).
Benzyl ester, cbz-L-leucyl monoester (21)
De-protection of the cyclopentenyl hydroxyl in the t-butyldimethysilyl
monoester 20 succeeded by treatment with diluted hydrochloric acid solution as
described for 18, with the exception that a 1:5 (v/v) chloroform-ethanol
mixture,
instead of ethanol alone, was used to ensure homogeneity. Work-up afforded 20,
colorless oil, in 87.6% yield.
L-leucyl monoesler (22)
Hydrogenolysis of the benzyl and N-carbobenzyloxy groups in 21 was carried
out as for 18. Work-up afforded 22 (95.3%), white solid, m.p. 118-120 C.
Synthesis of 2-L-leucyl ester of treprostinil 25
0
N-OOC= i H-CH2CH(CH2)2 OH
OH NH=COOCH2C6H5 OOC=CH(NHcbz)=CH2CH(CH3)2 +
ON O
OCH2000CH2O6H5
OCH2OOOCH2C6H5
23
13
93g.H C H(NHcbz)=CH2CH(CH3)2 OOC=CH(NHcbz)=CH2CH(CH3)2
OOC-CH(NHcbz)=CH2CH(CH3)2
OCH2000CH2C6H5 OCH2000CH2C6H5
21
24
29
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Benzyl ester, cbz-L-Ieucyl monoesters (21, 23) and -diester (24)
To a stirred solution of ester 13 (0.53 g: 1.10 mmol) and N-carbobenzyloxy-L-
leucine N-hydroxysuccinimide ester (0.76 g; 2.05 mmol) in dichioromethane (30
ml)
4-(dimethyamino) pyridine (0.29 g; 2.37 inmol) was added, then stirring
continued at
room temperature for 1 day. The solution was diluted with dichioromethane (40
mnl),
successively washed with a 5% sodium hydroxide solution (4 x 25 ml), 10%
hydrochloric acid (2 x 30 ml), 5% sodium bicarbonate solution (50 ml), dried
over
anhydrous sodium sulfate and concentrated under reduced pressure to give the
crude
product (0.85 g), as a viscous, yellow oil. Thin layer chromatography revealed
a
complex mixture in which esters 13 and 21 as well as cbz-L-leucine could be
identified through the corresponding rF values, only as minor products. The
crude
product was flash-chromatographed through a silica gel column eluting with
gradient
hexane-ethyl ether. At 7:3 (v/v) hexane-ethyl ether, the first fraction gave
the cbz-L-
leucyl diester 24 (6% of the product subjected to chromatography) while the
two
subsequent fractions afforded the cbz-L-leucyl monoester 23 (54% of the crude
product, as pure isolated 23; 57.6% yield, relative to 2). Purity of both
compounds
was verified by analytical tlc and NMR. The other isomer, cbz-L-leucyl
monoester
21 constituted only about 5% of the crude product and was isolated by
preparative tlc
of the latter only a 3:1 23/21 mixture.
t ,cFJ{ iH l y C ~ (Cl ~ l y 0czf~N1 2 . H2C CK
à {:I ~klz ~ OCII OiI
23 25
L-leucyl monoester (25)
Hydrogenolysis of 23 to the ester 25 was performed as described for
compound 12 but reaction was carried out at 35 p.s.i., overnight. Work-up and
drying
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
over phosphorus pentoxide in vacuo afforded 25, white solid m. p. 153-155 C,
in
quantitative yield.
Synthesis of 3'-L-alanyl ester of treprostinil 30
HO ,. -NO2
CH .CII Cf t1H C112.CRCOO.C6l pNO2
NRCOOCH2C6H5 DCC 1 H.COQCFI2C6H5
26 27
N-Cbz-L-alanyl p-nitro phenyl ester (27)
To a stirred solution containing N-carbobenzyloxy-L-alanine (1 g; 4.48 mmol)
and p-nitrophenol (1 g; 7.19 mmol) in anhydrous tetrahydrofuran (7 ml) a fine
suspension of 1,3-dicyclohexylcarbodiiinide (1.11 g; 5.38 minol) in
tetrahydrofuran (5
ml) was added over 30 min. Stirring was continued at room temperature for 18
hrs.,
glacial acetic acid (0.3 ml) added, 1,3-dicyclohexylurea filtered off and
solvent
removed in vacuo, at 40 C, to give a viscous, yellow-reddish oil (2.5 g). The
1H-
NMR spectrum showed a mixture consisting of N-carbobenzyloxy-Lalanine p-
nitrophenyl ester (27), unreacted p-nitrophenol and a small amount of DCU,
which
was used as such in the next reaction step.
OH CII .CII,Cot .C II . Qg OOC.CFI(NH'cbx).CH
c OTBDKIS NILCOOCIIIC, IIg ~- C~ ~6bh1
DMAP
OCHICOOCIIIC61j OCHICOOC112C H
16 28
31
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
COC,CH(NII6z).C11j "4, y..
1101 3H 1121 Pd s L -OH
FIOH
OCHCOUCH2C6H5 OCll COO H
2.. 30
Benzyl ester, cbz-L-alanyl monoester (29)
A solution of 4-(dimethylamino)pyridine (0.30 g; 2.49 mmol) in
dichloromethane (3 ml) was quickly dropped (over 5 min.) into a magnetically
stirred
solution of ester 16 (0.37 g; 0.62 mmol) and crude N-carbobenzyloxy-L-alanine
p-
nitrophenyl ester (0.98 g) in dichloromethane (12 ml). The mixture was stirred
overnight at room temperature, then diluted with dichloromethanc (50 ml), and
thoroughly washed with a 5% sodium hydroxide solution (7 x 35 ml), 10%
hydrochloric acid (3 x 35 ml), 5 /a sodium bicarbonate solution (50 ml), dried
over
anhydrous sodium sulfate and concentrated under reduced pressure to give the
crude
ester 28 (1.1 g). The latter was dissolved in ethanol (30 ml), 5% hydrochloric
acid (8
ml) and chloroform (5 ml) were added and the solution stirred overnight.
Solvents
were removed in vacuo, the residue taken-up in dichloromethane, washed to pH 7
with a 5% sodium hydrogencarbonate solution, dried over anhydrous sodium
sulfate
and the solvent evaporated affording crude 29 (1.04 g). Purification by column
chromatography on silica gel, eluting with gradient hexane-ethyl ether,
enabled
separation of a fraction (at hexane: ethyl ether = 1:1 v/v) of pure 29 as a
colorless very
viscous oil (0.11 g; 25.8% overall yield, based on 16).
L-alanyl monoester (30)
Removal of the benzyl and N-carbobenzyloxy groups in 29 was achieved
through catalytic hydrogenation as described for 12. Ester 30 was obtained
(yield:
97.2%) as a pale-yellow, partially crystallized, oil.
32
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Synthesis of the 3'-L-valine ester of Treprostinil benzyl ester 33
4H: H Il. C31-I
tH
~H CJ13C."1 12CI
03 -10H CI
C<H3GN }~ CFi I
rclW C}
C OOH 10CH2C4H5 )UC H2C&H5
1 13 16
N Cbz-Irval k DCC, DNW
Mch
rx
kI C:iC CIICII(CI13)2 00CCHCII(CH3)2
NHOOCCH2C6H5 NH( }CCH2,C FI5
.õwoH
"OTHDNIS
1OOCH2C6H COOCRIC HS
32 31
H2, Pd/C
1I OOCCISCH(CHH2
NI12
H
C
COON
33
Synthesis of the benzyl ester of Treprostinil 13
The benzyl ester 11 was synthesized by adapting the method described by J.
C. Lee et al. in Organic Prep. and Proc. Intl., 1996, 28(4), 480-483. To a
solution of 1
(620 ing, 1.6 mmoles) and cesium carbonate (782.4 mg, 2.4 mmoles) in
acetonitrile
(30 ml) was added benzyl bromide (0.48 ml, 4 mmoles) and the mixture was
stirred at
reflux for 1 hour. After cooling at room temperature, the precipitate was
filtered off
and the filtrate was concentrated in vacuo. The residue was dissolved in
chloroform
(150 ml) and washed with a 2% aqueous solution of NaHCO3 (3 x 30 ml). The
33
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
organic layer was washed with brine, dried on Na2SO4, filtered and the solvent
was
removed in vacuo to afford 750 mg of the crude benzyl ester 13 (yield 98%) as
a
yellow viscous oil. The crude benzyl ester 13 can be purified by column
chromatography (100-0% dichioromethane(methanol) but it can also be used crude
in
subsequent reactions.
Synthesis of the TBDMS protected Treprostinil benzyl ester 16
The procedure for the synthesis of the TBDMS protected benzyl ester was
adapted from Organic Synth., 1998, 75, 139-145. The benzyl ester 13 (679 mg,
1.4
mmoles) was dissolved in anhydrous dichloromethanc (20 ml) and the solution
was
cooled to 0 C on an ice bath. Imidazole (192 mg, 2.8 mmoles) and t-butyl-
dimethylsilyl chloride (TBDMSCI) (420 mg, 2.8 mmoles) were added and the
mixture was maintained under stirring for another half hour on the ice bath
and then it
was left overnight at room temperature. 40 ml water was added to the reaction
mixture and the organic layer was separated. The aqueous layer was extracted
with 3
x 50 ml dichloromethane. The combined organic layers were dried over Na2SO4,
filtered and the solvent was removed in vacuo. This afforded 795 mg of
material
which proved to be a mixture of the desired mono TBDMS protected 5 benzyl
ester
with the bis-TBDMS protected benzyl ester. Pure 16 (249 mg) was obtained by
column chromatography on silica gel (eluent 35% ethyl acetate/hexane).
Synthesis of N-Cbz-L-valine ester of the TBDMS protected Treprostinil benzyl
ester 31
The procedure used was adapted from Tetrahedron Lett., 1978, 46, 4475-4478.
A solution of NCbz-L-valine (127 mg, 0.5 mmoles), N,N-dicyclohexylcarbodiimide
(DCC) (111 mg, 0.5 mmoles), compound 16 (249 mg, 0.4 mmoles) and 4-
(dimethylamino)pyridine (DMAP) (6 mg, 0.05 mmoles) in anhydrous
dichloromethane (15 ml) was stirred at room temperature until esterification
was
complete. The solution was filtered and the formed N,N-dicyclohexylurea was
filtered. The filtrate was diluted with dichloromethane (80 ml) and washed
with water
34
CA 02526534 2010-01-11
WO 2005/007081 PCT/US2004/016401
(3 x 30 ml), a 5% aqueous acetic acid solution (2 x 30 ml) and then again with
water
(3 x 30 ml). The organic layer was dried over Na2SO4 and the solvent was -
evaporated
in vacuo affording 369 mg crude 31. Pure 31 was obtained by chromatography
(silica
gel, 35% ethyl acetate/hexane).
Synthesis of the 3'-N-Cbz-L-valine ester of Treprostinil benzyl ester 32
Cleavage of the TBDMS group in compound 31 was achieved using an
adaptation of the procedure described in Org. Letters, 2000, 2(26), 4177-4180.
The
N-Cbz-L-valine ester of the TBDMS protected benzyl ester 31 (33 mg, 0.04
mmoles)
was dissolved in methanol (5 ml) and tetrabutylammonium tribromide (TBATB) (2
mg, 0.004 mmoles) was added. The reaction mixture was stirred at room
temperature
for 24 his until the TBDMS deprotection was complete. The methanol was
evaporated and the residue was taken in dichloromethane. The dichloromethane
solution was washed with brine and then dried over Na2SO4. After filtering the
drying agent the solvent was evaporated to dryness affording 30.2 mg of crude
compound 32.
Synthesis of the 3'-L-valinc ester of Treprostinil 33
The benzyl and benzyl carboxy groups were removed by catalytic
hydrogenation at atmospheric pressure in the presence of palladium 10% wt on
activated carbon. The 3'-N-Cbz-L-valine ester of benzyl ester 32 (30.2 mg,
0.04
mmoles) was dissolved in methanol (10 ml) and a catalytic amount of Pd/C was
added. Under magnetic stirring the air was removed from the flask and then
hydrogen
was admitted. The reaction mixture was maintained under hydrogen and stirring
at
room temperature for 24 his, then the hydrogen was removed with vacuum. The
reaction mixture was then filtered through a layer of celit~r and the solvent
was
removed in vacua to afford the pure 3'-L-valine ester of Treprostinil 33 (15
mg, 0.03
mmoles).
* Trade-mark
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Synthesis of 2-L-valine ester of Treprostinil 36/ 'bis-L-valine ester of
Trenrostinil
37
Synthesis of 2-L-alanine ester of Treprostinil 36'/ bis-L-alanine ester of
Treprostinil 37'
H. H
ccH2Cl
+knltl Ca~C, .ia61#SFE
CHjCN
a ~Si[3Li7S.
OH
COOll (OOCH2C6Hs R-CH-HCIz
DCC, T~ N
CIhCI
1 13
H
H 60C UR OH
1 ICbz W10MCHR
D CC II + IIIC a
'ICOCH2C6H5
COOCH2C6H$
35 R _ CH(CH3)2 34 R CH(C )2
3'R=CH3 34" b=CHI
H2,PdAC 142, Pd)C
McOH MeOH
OOCCHR
ON.
I
0 CCCIlIt~ I f
N'IZ
COOH COOH
37 R CH(CH311 36 R = CH(CH ')
37'R=CH3 36'R=C.H3
36
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Synthesis of 2-N-Cbz-L-valine ester of Treprostinil benzyl ester 34 and bis-N-
Cbz-L-valine ester of Treprostinil benzyl ester 35
The procedure used was adapted from Tetrahedron Lett., 1978, 46, 4475-4478.
A solution of NCbz-L-valine (186 mg, 0.7 mmoles), N,N-dicyclohexylcarbodiimide
(DCC) (167 mg, 0.8 mmoles), compound 13 (367 mg, 0.8 mmoles) and 4-
(dimethylamino)pyridine (DMAP) (12 mg, 0.09 imnoles) in anhydrous
dichloromethane (15 ml) was stirred at room temperature until esterification
was
complete. The solution was filtered and the formed N,N-dicyclohexylurea was
filtered. The filtrate was diluted with dichloromethane (100 ml) and washed
with
water (3 x 50 ml), a 5% aqueous acetic acid solution (2 x 50 ml) and then
again with
water (3 x 50 ml). The organic layer was dried over Na2SO4 and the solvent was
evaporated in vacuo affording 556 mg crude product. The product was separated
by
chromatography (silica gel, 35% ethyl acetate/hexane) yielding 369.4 mg 2-
valine ester
34 and 98 mg bis-valine ester 35.
Synthesis of 2 N-Cbz-L-alanine ester of Treprostinil benzyl ester 34' and bis-
N-
Cbz-L-alanine ester of Treprostinil benzyl ester 35'
The procedure used was adapted from Tetrahedron Lett., 1978, 46, 4475-4478.
A solution of NCbz-L-alanine (187 mg, 0.84 mmoles), N,N-
dicyclohexylcarbodiimide
(DCC) (175 mg, 0.85 mmoles), compound 13 (401 mg, 0.84 mmoles) and 4-
(dimethylamino)pyridine (UMAP) (11.8 mg, 0.1mmoles) in anhydrous
dichloromethane (15 ml) was stirred at room temperature until esterification
was
complete. The solution was filtered and the formed N,N-dicyclohexylurea was
filtered. The filtrate was diluted with dichloromethane (100 ml) and washed
with
water (3 x 50 ml), a 5% aqueous acetic acid solution (2 x 50 ml) and then
again with
water (3 x 50 ml). The organic layer was dried over Na2SO4 and the solvent was
evaporated in vacuo affording 516 mg crude product. The product was separated
by
chromatography (silica gel, 35% ethyl acetate/hexane) yielding 93.4 mg 2-
alanine ester
34' and 227 mg bis-alanine ester 35'.
37
CA 02526534 2010-01-11
WO 2005/007081 PCT/US2004/016401
Synthesis of 2-L-valine ester of Treprostinil 36/ bis-L-valine ester of
Treprostinil
37
The benzyl and benzyl carboxy groups were removed by catalytic
hydrogenation at atmospheric pressure in the presence of palladium 10% wt on
activated carbon. The 2-N-Cbz-L-valine ester of Treprostinil benzyl ester 34
(58.2
mg, 0.08 mmoles) / bis-N-Cbz-L-valine ester of Treprostinil benzyl ester 35
(55.1 mg,
0.06 mmoles) was dissolved in methanol (10 nil) and a catalytic amount of Pd/C
was
added. Under magnetic stirring the air was removed from the flask and hydrogen
was
admitted. The reaction mixture was maintained under hydrogen and stirring at
room
temperature for 20 hrs, then hydrogen was removed with vacuum. The reaction
mixture was then filtered through a layer of celite and the solvent was
removed in
vacuo to afford the pure 2-L-valine ester of Treprostinil 36 (40 mg, 0.078
nnmoles)/
bis-L-valine ester of Treprostinil 37 (23 nag, 0.04 mmoles).
Synthesis of 2-L-alanine ester of Treprostinil 36'/ bis-L-alanine ester of
Treprostinil 37'
The benzyl and benzyl carboxy groups were removed by catalytic
hydrogenation at atmospheric pressure in the presence of palladium 10% wt on
activated carbon. The 2-N-Cbz-L-alanine ester of Treprostinil benzyl ester 34'
{87.4
mg, 0.13 mmoles) / bis-N-Cbz-L-alanine ester of Treprostinil benzyl ester 35'
(135
mg, 0.15 mmoles) was dissolved in methanol (15 ml) and a catalytic amount of
Pd/C
was added. Under magnetic stirring the air was removed from the flask and
hydrogen
was admitted. The reaction mixture was maintained under hydrogen and stirring
at
room temperature for 20 hrs, then hydrogen was removed with vacuum. The
reaction
mixture was then filtered through a layer of celite and the solvent was
removed in
vacuo to afford the pure 2-L-valine ester of Treprostinil 36' (57 mg, 0.12
mmoles)/
bis-L-alanine ester of Treprostinil 37' (82 mg, 0.15 mmoles).
* Trade-mark
3B
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Synthesis of benzyl esters of treprostinil 38 a-e
OH
IOH
At 13r, TFA I
O
38
a 4-NO2C6H4CH2; b 4-(CH3O)C6H4CH2i c 2-C1C6H4CH2; d 2,4-(NO2)2C6H3CH2i e 4-
FG6H4CH2 Synthesis of the benzyl esters of treprostinil 38 a-e was performed
using
the procedure for the benzyl ester 13.
Enantiomers of these compounds, shown below, can be synthesized using
reagents and synthons of enantiomeric chirality of the above reagents.
H
OR3
OR2
H
OCH2CO2R1
(-)-treprostinil can be synthesized as follows:
39
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
O RO
OCH OTHP
OCH3
3 3
bE~ 6aR=H
C 6b R= TBDMS
TBDMSO
H_
9ThP9 O
H II
OCH3 OCH3
7
le
1L H = H
6R1 h OR
ORZ o R
H H
OCH3 OCH3 10a R=p-NO2C6H4CO
f[:~9a R1= THP, R,=H 10b R=H
9b R3= THP, R2=Bn
gF 9c R1= H, R2=Bn
H - 1 H
OH
OH OH
PCL- OH YCn__>
OH 11a R== CH3
11-1 CO2H 2 11b R=H
(a) (S)-2-methyl-CBS-oxazaborolidine, BH3=SMe2, THF, -30 C, 85%. (b)
TBDMSCI, imidazole, CH2C12, 95%. (c) Co2(CO)8, CH2C12, 2hr. r.t., then CH3CN,
2hr. reflux. 98%. (d) K2C03, Pd/C (10%), EtOH, 50 psi/24 hr. 78% (e) NaOH,
EtOH,
NaBH4. 95%. (f) BnBr, NaH, THF, 98%. (g).CH3OH, TsOH. 96%. (h) i. p-
nitrobenzoic acid, DEAD, TPP,benzene. (i) CH3OH, KOH. 94%. (j) Pd/C (10%),
EtOH, 50 psi/2 hr. quant. (k). Ph2PLi, THF. (1) i. C1CH2CN, K2C03. ii, KOH,
CH3OH, reflux. 83 % (2 steps).
Briefly, the enantiomer of the commercial drug (+)-Treprostinil was
synthesized using the stereoselective intramolecular Pauson I,'-hand reaction
as a key
step and Mitsunobu inversion of the side-chain hydroxyl group. The absolute
configuration of (-)-Treprostinil was confirmed by an X-ray structure of the L-
valine
amide derivative.
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
The following procedure was used to make (-)-treprostinil-methyl-L-valine
amide: To a stirred solution of (-)-Treprostinil (391mg, lmmol) and L-valine
methyl
ester hydrochloride (184 mg, 1.1 mmol) in DMF (1 Oml) under Ar was
sequentially
added pyBOP reagent (1.04g, 2mmol), diisopropylethyl amine (0.52ml, 3mmol).
The
reaction mixture was stirred at room temperature overnight (15hrs). Removal of
the
solvent in vacuo and purification by chromatography yielded white solid 12
(481mg,
86%), which was recrystallized (10% ethyl acetate in hexane) to give suitable
crystals
for X-ray.
Various modifications of these synthetic schemes capable of producing
additional compounds discussed herein will be readily apparent to one skilled
in the
art.
There are two major barriers to deliver treprostinil in the circulatory
system.
One of these barriers is that treprostinil undergoes a large first pass
effect. Upon first
circulating through the liver, about 60% of treprostinil plasma levels are
metabolized,
which leaves only about 40% of the absorbed dose. Also, a major barrier to
oral
delivery for treprostinil is that the compound is susceptible to an efflux
mechanism in
the gastrointestinal tract. The permeability of treprostinil has been measured
across
Caco-2 cell monolayers. The apical to basal transport rate was measured to be
1.39 X
106 cm/sec, which is indicative of a highly permeable compound. However, the
basal
to apical transport rate was 12.3 X 106 cm/sec, which suggests that
treprostinil is
efficiently effluxed from the serosal to lumenal side of the epithelial cell.
These data
suggest that treprostinil is susceptible to p-glycoprotein, a membrane bound
multidrug
transporter. It is believed that the p-glycoprotein efflux pump prevents
certain
pharmaceutical compounds from traversing the mucosal cells of the small
intestine
and, therefore, from being absorbed into systemic circulation.
Accordingly, the present invention provides pharmaceutical compositions
comprising treprostinil, the compound of structure I or the compound of
structure II,
or their pharmaceutically acceptable salts and combinations thereof in
combination
with one or more inhibitors of p-glycoprotein. A number of known non-cytotoxic
pharmacological agents have been shown to inhibit p-glycoprotein are disclosed
in
U.S. Patent Nos. 6,451,815, 6,469,022, and 6,171,786.
41
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
P-glycoprotein inhibitors include water soluble forms of vitamin E,
polyethylene glycol, poloxamers including Pluronic F-68, polyethylene oxide,
polyoxyethylene castor oil derivatives including Cremophor EL and Cremophor RH
40, Chrysin, (+)-Taxifolin, Naringenin, Diosmin, Quercetin, cyclosporin A
(also
known as cyclosporine), verapamil, tamoxifen, quinidine, phenothiazines, and
9,10-
dihydro-5-methoxy-9-oxo-N-[4-[2-(1,2,3,4-tetrahydro-6,7,-dimethoxy-2-
isoquinolinyl)ethyl]phenyl]-4-acridinecarboxamide or a salt thereof.
Polyethylene glycols (PEGs) are liquid and solid polymers of the general
formula H(OCH2CH2)õOH, where n is greater than or equal to 4, having various
average molecular weights ranging from about 200 to about 20,000. PEGs are
also
known as alpha-hydro-omega-hydroxypoly-(oxy-1,2-ethanediyl)polyethylene
glycols.
For example, PEG 200 is a polyethylene glycol wherein the average value of n
is 4
and the average molecular weight is from about 190 to about 210. PEG 400 is a
polyethylene glycol wherein the average value of n is between 8.2 and 9.1 and
the
average molecular weight is from about 380 to about 420. Likewise, PEG 600,
PEG
1500 and PEG 4000 have average values of n of 12.5-13.9, 29-36 and 68-84,
respectively, and average molecular weights of 570-630, 1300-1600 and 3000-
3700,
respectively, and PEG 1000, PEG 6000 and PEG 8000 have average molecular
weights of 950-1050, 5400-6600, and 7000-9000, respectively. Polyethylene
glycols
of varying average molecular weight of from 200 to 20000 are well known and
appreciated in the art of pharmaceutical science and are readily available.
The preferred polyethylene glycols for use in the instant invention are
polyethylene glycols having an average molecular weight of from about 200 to
about
20,000. The more preferred polyethylene glycols have an average molecular
weight
of from about 200 to about 8000. More specifically, the more preferred
polyethylene
glycols for use in the present invention are PEG 200, PEG 400, PEG 600, PEG
1000,
PEG 1450, PEG 1500, PEG 4000, PEG 4600, and PEG 8000. The most preferred
polyethylene glycols for use in the instant invention is PEG 400, PEG 1000,
PEG
1450, PEG 4600 and PEG 8000.
Polysorbate 80 is an oleate ester of sorbitol and its anhydrides copolymerized
with approximately 20 moles of ethylene oxide for each mole of sorbitol and
sorbitol
42
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
anhydrides. Polysorbate 80 is made up of sorbitan mono-9-octadecanoate
poly(oxy-
1,2-ethandiyl) derivatives. Polysorbate 80, also known as Tween 80, is well
known
and appreciated in the pharmaceutical arts and is readily available.
Water-soluble vitamin E, also known as d-alpha-tocopheryl polyethylene
glycol 1000 succinate [TPGS], is a water-soluble derivative of natural-source
vitamin
E. TPGS may be prepared by the esterification of the acid group of crystalline
d-
alpha-tocopheryl acid succinate by polyethylene glycol 1000. This product is
well
known and appreciated in the pharmaceutical arts and is readily available. For
example, a water-soluble vitamin E product is available commercially from
Eastman
Corporation as Vitamin E TPGS.
Naringenin is the bioflavonoid compound 2,3-dihydro-5,7-dihydroxy-2-(4-
hydroxyphenyl) -4H-1-benzopyran-4-one and is also known as 4',5,7-
trihydroxyflavanone. Naringenin is the aglucon of naringen which is a natural
product found in the fruit and rind of grapefruit. Naringenin is readily
available to the
public from commercial sources.
Quercetin is the bioflavonoid compound 2-(3,4-dihydroxyphenyl)-3,5,7-
trihydroxy -4H-1-benzopyran-4-one and is also known as 3,3',4',5,7-
pentahydroxyflavone. Quercetin is the aglucon of quercitrin, of rutin and of
other
glycosides. Quercetin is readily available to the public from commercial
sources.
Diosmin is the naturally occurring flavonic glycoside compound 7-[[6- -6-
deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranosyl] oxy] -5-hydroxy-2-(3-
hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one. Diosmin can be isolated from
various plant sources including citrus fruits. Diosmin is readily available to
the public
from commercial sources.
Chrysin is the naturally occurring compound 5,7-dihydroxy-2-phenyl-4H-1-
benzopyran-4-one which can be isolated from various plant sources. Chrysin is
readily available to the public from commercial sources.
Poloxamers are alpha-hydro-omega-hydroxypoly(oxyethylene)poly
(oxypropylene)poly(oxyethylene) block copolymers. Poloxamers are a series of
closely related block copolymers of ethylene oxide and propylene oxide
conforming
43
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
to the general formula HO(C2H4O)a(C3H60)b(C2H4 O)aH. For example, poloxamer
124 is a liquid with "a" being 12, "b" being 20, and having an average
molecular
weight of from about 2090 to about 2360; poloxamer 188 is a solid with "a"
being 80,
"b" being 27, and having an average molecular weight of from about 7680 to
about
9510; poloxamer 237 is a solid with "a" being 64, "b" being 37, and having an
average molecular weight of from about 6840 to about 8830; poloxamer 338 is a
solid
with "a" being 141, "b" being 44, and having an average molecular weight of
from
about 12700 to about 17400; and poloxamer 407 is a solid with "a" being 101,
"b"
being 56, and having an average molecular weight of from about 9840 to about
14600. Poloxamers are well known and appreciated in the pharmaceutical arts
and
are readily available commercially. For example, Pluronic F-68 is a
commercially
available poloxamer from BASF Corp. The preferred poloxamers for use in the
present invention are those such as poloxamer 188, Pluronic F-68, and the
like.
Polyoxyethylene castor oil derivatives are a series of materials obtained by
reacting varying amounts of ethylene oxide with either castor oil or
hydrogenated
castor oil. These polyoxyethylene castor oil derivatives are well known and
appreciated in the pharmaceutical arts and several different types of material
are
commercially available, including the Cremophors available from BASF
Corporation.
Polyoxyethylene castor oil derivatives are complex mixtures of various
hydrophobic
and hydrophilic components. For example, in polyoxyl 35 castor oil (also known
as
Cremophor EL), the hydrophobic constituents comprise about 83% of the total
mixture, the main component being glycerol polyethylene glycol ricinoleate.
Other
hydrophobic constituents include fatty acid esters of polyethylene glycol
along with
some unchanged castor oil. The hydrophilic part of polyoxyl 35 castor oil
(17%)
consists of polyethylene glycols and glyceryl ethoxylates.
In polyoxyl 40 hydrogenated castor oil (Cremophor RH 40) approximately
75% of the components of the mixture are hydrophobic. These comprise mainly
fatty
acid esters of glycerol polyethylene glycol and fatty acid esters of
polyethylene
glycol. The hydrophilic portion consists of polyethylene glycols and glycerol
ethoxylates. The preferred polyoxyethylene castor oil derivatives for use in
the
present invention are polyoxyl 35 castor oil, such as Cremophor EL, and
polyoxyl 40
44
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
hydrogenated castor oil, such as Cremophor RH 40. Cremophor EL and Cremophor
RH 40 are commercially available from BASF Corporation.
Polyethylene oxide is a nonionic homopolymer of ethylene oxide conforming
to the general formula (OCH2CH2)õ in which n represents the average number of
oxyethylene groups. Polyethylene oxides are available in various grades which
are
well known and appreciated by those in the pharmaceutical arts and several
different
types of material are commercially available. The preferred grade of
polyethylene
oxide is NF and the like which are commercially available.
(+)-Taxifolin is (2R-trans)-2-(3,4-dihydroxyphenyl)-2,3-dihydro-3,5,7-
trihydroxy-4H-1-benzo pyran-4-one. Other common names for (+)-taxifolin are
(+)-
dihydroquercetin; 3,3', 4', 5,7-pentahydroxy-flavanone; diquertin; taxifoliol;
and
distylin. (+)-Taxifolin is well know and appreciated in the art of
pharmaceutical arts
and is readily available commercially.
The preferred p-glycoprotein inhibitor for use in the present invention are
water soluble vitamin E, such as vitamin E TPGS, and the polyethylene glycols.
Of
the polyethylene glycols, the most preferred p-glycoprotein inhibitors are PEG
400,
PEG 1000, PEG 1450, PEG 4600 and PEG 8000.
Administration of a p-glycoprotein inhibitor may be by any route by which the
p-glycoprotein inhibitor will be bioavailable in effective amounts including
oral and
parenteral routes. Although oral administration is preferred, the p-
glycoprotein
inhibitors may also be administered intravenously, topically, subcutaneously,
intranasally, rectally, intramuscularly, or by other parenteral routes. When
administered orally, the p-glycoprotein inhibitor may be administered in any
convenient dosage form including, for example, capsule, tablet, liquid,
suspension,
and the like.
Generally, an effective p-glycoprotein inhibiting amount of a p-glycoprotein
inhibitor is that amount which is effective in providing inhibition of the
activity of the
p-glycoprotein mediated active transport system present in the gut. An
effective p-
glycoprotein inhibiting amount can vary between about 5 mg to about 1000 mg of
p-
glycoprotein inhibitor as a daily dose depending upon the particular p-
glycoprotein
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
inhibitor selected, the species of patient to be treated, the dosage regimen,
and other
factors which are all well within the abilities of one of ordinary skill in
the medical
arts to evaluate and assess. A preferred amount however will typically be from
about
50 ing to about 500 mg, and a more preferred amount will typically be from
about 100
mg to about 500 mg. The above amounts of a p-glycoprotein inhibitor can be
administered from once to multiple times per day. Typically for oral dosing,
doses
will be administered on a regimen requiring one, two or three doses per day.
Where water soluble vitamin E or a polyethylene glycol is selected as the p-
glycoprotein inhibitor, a preferred amount will typically be from about 5 mg
to about
1000 mg, a more preferred amount will typically be from about 50 mg to about
500
mg, and a further preferred amount will typically be from about 100 mg to
about 500
mg. The most preferred amount of water soluble vitamin E or a polyethylene
glycol
will be from about 200 mg to about 500 mg. The above amounts of water soluble
vitamin E or polyethylene glycol can be administered from once to multiple
times per
day. Typically, doses will be administered on a regimen requiring one, two or
three
doses per day with one and two being preferred.
As used herein, the term "co-administration" refers to administration to a
patient of both a compound that has vasodilating, and/or platelet aggregation
inhibiting
properties, including the compounds described in U.S. Patent Nos. 4,306,075
and
5,153,222 which include treprostinil and structures I and II described herein,
and a p-
glycoprotein inhibitor so that the pharmacologic effect of the p-glycoprotein
inhibitor
in inhibiting p-glycoprotein mediated transport in the gut is manifest at the
time at
which the compound is being absorbed from the gut. Of course, the compound and
the p-glycoprotein inhibitor may be administered at different times or
concurrently.
For example, the p-glycoprotein inhibitor may be administered to the patient
at a time
prior to administration of the therapeutic compound so as to pre-treat the
patient in
preparation for dosing with the vasodilating compound. Furthermore, it may be
convenient for a patient to be pre-treated with the p-glycoprotein inhibitor
so as to
achieve steady state levels of p-glycoprotein inhibitor prior to
administration of the
first dose of the therapeutic compound. It is also contemplated that the
vasodilating
and/or platelet aggregation inhibiting compounds and the p-glycoprotein
inhibitor
46
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
may be administered essentially concurrently either in separate dosage forms
or in the
same oral dosage form.
The present invention further provides that the vasodilating and/or platelet
aggregation inhibiting compound and the p-glycoprotein inhibitor may be
administered in separate dosage forms or in the same combination oral dosage
form.
Co-administration of the compound and the p-glycoprotein inhibitor may
conveniently be accomplished by oral administration of a combination dosage
form
containing both the compound and the p-glycoprotein inhibitor.
Thus, an additional embodiment of the present invention is a combination
pharmaceutical composition for oral administration comprising an effective
vasodilating and/or platelet aggregation inhibiting amount of a compound
described
herein and an effective p-glycoprotein inhibiting amount of a p-glycoprotein
inhibitor.
This combination oral dosage form may provide for immediate release of both
the
vasodilating and/or platelet aggregation inhibiting compound and the p-
glycoprotein
inhibitor or may provide for sustained release of one or both of the
vasodilating and/or
platelet aggregation inhibiting compound and the p-glycoprotein inhibitor. One
skilled in the art would readily be able to determine the appropriate
properties of the
combination dosage form so as to achieve the desired effect of co-
administration of
the vasodilating and/or platelet aggregation inhibiting compound and the p-
glycoprotein inhibitor.
Accordingly, the present invention provides for an enhancement of the
bioavailability of treprostinil, a drug of structure I or II, and
pharmaceutically
acceptable salts thereof by co-administration of a p-glycoprotein inhibitor.
By co-
administration of these compounds and a p-glycoprotein inhibitor, the total
amount of
the compound can be increased over that which would otherwise circulate in the
blood in the absence of the p-glycoprotein inhibitor. Thus, co-administration
in
accordance with the present invention can cause an increase in the AUC of the
present
compounds over that seen with administration of the compounds alone.
Typically, bioavailability is assessed by measuring the drug concentration in
the blood at various points of time after administration of the drug and then
47
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
integrating the values obtained over time to yield the total amount of drug
circulating
in the blood. This measurement, called the Area Under the Curve (AUC), is a
direct
measurement of the bioavailability of the drug.
Without limiting the scope of the invention, it is believed that in some
embodiments derivatizing treprostinil at the R2 and R3 hydroxyl groups can
help
overcome the barriers to oral treprostinil delivery by blocking these sites,
and thus the
metabolism rate may be reduced to permit the compound to bypass some of the
first
pass effect. Also, with an exposed amino acid, the prodrug may be actively
absorbed
from the dipeptide transporter system that exists in the gastrointestinal
tract.
Accordingly, the present invention provides compounds, such as those found in
structures I and II, that reduce the first pass effect of treprostinil and/or
reduce the
efflux mechanism of the gastrointestinal tract.
In some embodiments of the method of treating hypertension in a subject, the
subject is a mammal, and in some embodiments is a human.
Pharmaceutical formulations may include any of the compounds of any of the
embodiments described above, either alone or in combination, in combination
with a
pharmaceutically acceptable carrier such as those described herein.
The instant invention also provides for compositions which may be prepared
by mixing one or more compounds of the instant invention, or pharmaceutically
acceptable salts thereof, with pharmaceutically acceptable carriers,
excipients,
binders, diluents or the like, to treat or ameliorate a variety of disorders
related
vasoconstriction and/or platelet aggregation. A therapeutically effective dose
further
refers to that amount of one or more compounds of the instant invention
sufficient to
result in amelioration of symptoms of the disorder. The pharmaceutical
compositions
of the instant invention can be manufactured by methods well known in the art
such
as conventional granulating, mixing, dissolving, encapsulating, lyophilizing,
emulsifying or levigating processes, among others. The compositions can be in
the
form of, for example, granules, powders, tablets, capsules, syrup,
suppositories,
injections, emulsions, elixirs, suspensions or solutions. The instant
compositions can
be formulated for various routes of administration, for example, by oral
administration,
48
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
by transmucosal administration, by rectal administration, transdermal or
subcutaneous
administration as well as intrathecal, intravenous, intramuscular,
intraperitoneal,
intranasal, intraocular or intraventricular injection. The compound or
compounds of the
instant invention can also be administered by any of the above routes, for
example in a
local rather than a systemic fashion, such as injection as a sustained release
formulation.
The following dosage forms are given by way of example and should not be
construed
as limiting the instant invention.
For oral, buccal, and sublingual administration, powders, suspensions,
granules,
tablets, pills, capsules, gelcaps, and caplets are acceptable as solid dosage
forms. These
can be prepared, for example, by mixing one or more compounds of the instant
invention, or pharmaceutically acceptable salts thereof, with at least one
additive or
excipient such as a starch or other additive. Suitable additives or excipients
are sucrose,
lactose, cellulose sugar, mamutol, maltitol, dextran, sorbitol, starch, agar,
alginates,
chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens,
casein,
albumin, synthetic or semi-synthetic polymers or glycerides, methyl cellulose,
hydroxypropylmethyl-cellulose, and/or polyvinylpyrrolidone . Optionally, oral
dosage
forms can contain other ingredients to aid in administration, such as an
inactive diluent,
or lubricants such'as magnesium stearate, or preservatives such as paraben or
sorbic acid,
or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a
disintegrating agent,
binders, thickeners, buffers, sweeteners, flavoring agents or perfuming
agents.
Additionally, dyestuffs or pigments may be added for identification. Tablets
may be
further treated with suitable coating materials known in the art.
Additionally, tests have shown that the present compounds, including
treprostinil, and in particular the compounds of structure I and II have
increased
bioavailability when delivered to the duodenum. Accordingly, one embodiment of
the
present invention involves preferential delivery of the desired compound to
the
duodenum as well as pharmaceutical formulations that achieve duodenal
delivery.
Duodenal administration can be achieved by any means known in the art. In one
of
these embodiments, the present compounds can be formulated in an enteric-
coated
dosage form. Generally, enteric-coated dosage forms are usually coated with a
polymer
that is not soluble at low pH, but dissolves quickly when exposed to pH
conditions of 3
49
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
or above. This delivery form takes advantage of the difference in pH between
the
stomach, which is about 1 to 2, and the duodenum, where the pH tends to be
greater than
4.
Liquid dosage forms for oral administration may be in the form of
pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, slurries
and
solutions, which may contain an inactive diluent, such as water.
Pharmaceutical
formulations may be prepared as liquid suspensions or solutions using a
sterile liquid,
such as, but not limited to, an oil, water, an alcohol, and combinations of
these.
Pharmaceutically suitable surfactants, suspending agents, emulsifying agents,
may be
added for oral or parenteral administration.
As noted above, suspensions may include oils. Such oil include, but are not
limited to, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil.
Suspension
preparation may also contain esters of fatty acids such as ethyl oleate,
isopropyl
myristate, fatty acid glycerides and acetylated fatty acid glycerides.
Suspension
formulations may include alcohols, such as, but not limited to, ethanol,
isopropyl
alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as but
not
limited to, poly(ethyleneglycol), petroleum hydrocarbons such as mineral oil
and
petrolatum; and water may also be used in suspension formulations.
Injectable dosage forms generally include aqueous suspensions or oil
suspensions which may be prepared using a suitable dispersant or wetting agent
and a
suspending agent. Injectable forms maybe in solution phase or in the form of a
suspension, which is prepared with a solvent or diluent. Acceptable solvents
or
vehicles include sterilized water, Ringer's solution, or an isotonic aqueous
saline
solution. Alternatively, sterile oils may be employed as solvents or
suspending
agents. Preferably, the oil or fatty acid is non-volatile, including natural
or synthetic
oils, fatty acids, mono-, di- or tri-glycerides.
For injection, the pharmaceutical formulation may be a powder suitable for
reconstitution with an appropriate solution as described above. Examples of
these
include, but are not limited to, freeze dried, rotary dried or spray dried
powders,
amorphous powders, granules, precipitates, or particulates. For injection, the
CA 02526534 2010-01-11
WO 2005/007051 PCT/US2004/016401
formulations may optionally contain stabilizers, pH modifiers, surfactants,
bioavailability modifiers and combinations of these. The compounds may be
formulated for parenteral administration by injection such as by bolus
injection or
continuous infusion. A unit dosage form for injection may be in ampoules or in
multi-dose containers.
Besides those representative dosage forms described above, pharmaceutically
acceptable excipients and carries are generally known to those skilled in the
art and
are thus included in the instant invention. Such excipients and carriers are
described,
for example, in "Rerningtons Pharmaceutical Sciences" Mack Pub. Co., New
Jersey
(1991).
The formulations of the invention may be designed for to be short-acting, fast-
releasing, long-acting, and sustained-releasing as descri
bed below. Thus, the
pharmaceutical formulations may also be formulated for controlled release or
for slow
release.
The instant compositions may also comprise, for example, micelles or
liposomes, or some other encapsulated fonn, or may be administered in an
extended
release form to provide a prolonged storage and/or delivery effect. Therefore,
the
pharmaceutical formulations may be compressed into pellets or cylinders and
implanted intramuscularly or subcutaneously as depot injections or as implants
such
as stents. Such implants may employ known inert materials such as silicones
and
biodegradable polymers.
Specific dosages may be adjusted depending on conditions of disease, the age,
body weight, general health conditions, sex, and diet of the subject, dose
intervals,
administration routes, excretion rate, and combinations of drugs. Any of the
above
dosage forms containing effective amounts are well within the bounds of
routine
experimentation and therefore, well within the scope of the instant invention.
A therapeutically effective dose may vary depending upon the route of
administration and dosage form. The preferred compound or compounds of the
instant invention is a formulation that exhibits a high therapeutic index. The
therapeutic index is the dose ratio between toxic and therapeutic effects
which can be
51
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
expressed as the ratio between LD50 and ED50. The LD50 is the dose lethal to
50% of
the population and the ED50 is the dose therapeutically effective in 50% of
the
population. The LD50 and ED50 are determined by standard pharmaceutical
procedures in animal cell cultures or experimental animals.
A method of preparing pharmaceutical formulations includes mixing any of
the above-described compounds with a pharmaceutically acceptable carrier and
water
or an aqueous solution.
Pharmaceutical formulations and medicaments according to the invention
include any of the compounds of any of the embodiments of compound of
structure I,
II or pharmaceutically acceptable salts thereof described above in combination
with a
pharmaceutically acceptable carrier. Thus, the compounds of the invention may
be
used to prepare medicaments and pharmaceutical formulations. In some such
embodiments, the medicaments and pharmaceutical formulations comprise any of
the
compounds of any of the embodiments of the compounds of structure I or
pharmaceutically acceptable salts thereof. The invention also provides for the
use of
any of the compounds of any of the embodiments of the compounds of structure
I, II
or pharmaceutically acceptable salts thereof for prostacyclin-like effects.
The
invention also provides for the use of any of the compounds of any of the
embodiments of the compounds of structure 1, II or pharmaceutically acceptable
salts
thereof or for the treatment of pulmonary hypertension.
The invention also pertains to kits comprising one or more of the compounds
of structure I or II along with instructions for use of the compounds. In
another
embodiment, kits having compounds with prostacyclin-like effects described
herein in
combination with one or more p-glycoprotein inhibitors is provided along with
instructions for using the kit.
By way of illustration, a kit of the invention may include one or more
tablets,
capsules, caplets, gelcaps or liquid formulations containing the bioenhancer
of the
present invention, and one or more tablets, capsules, caplets, gelcaps or
liquid
formulations containing a prostacyclin-like effect compound described herein
in
dosage amounts within the ranges described above. Such kits may be used in
52
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
hospitals, clinics, physician's offices or in patients' homes to facilitate
the co-
administration of the enhancing and target agents. The kits should also
include as an
insert printed dosing information for the co-administration of the enhancing
and target
agents.
The following abbreviations and definitions are used throughout this
application:
Generally, reference to a certain element such as hydrogen or H is meant to
include all isotopes of that element. For example, if an R group is defined to
include
hydrogen or H, it also includes deuterium and tritium.
As used herein, the term "p-glycoprotein inhibitor" refers to organic
compounds which inhibit the activity of the p-glycoprotein mediated active
transport
system present in the gut. This transport system actively transports drugs
which have
been absorbed from the intestinal lumen and into the gut epithelium back out
into the
lumen. Inhibition of this p-glycoprotein mediated active transport system will
cause
less drug to be transported back into the lumen and will thus increase the net
drug
transport across the gut epithelium and will increase the amount of drug
ultimately
available in the blood.
The phrases "oral bioavailability" and "bioavailability upon oral
administration" as used herein refer to the systemic availability (i.e.,
blood/plasma
levels) of a given amount of drug administered orally to a patient.
The phrase "unsubstituted alkyl" refers to alkyl groups that do not contain
heteroatoms. Thus the phrase includes straight chain alkyl groups such as
methyl,
ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,
dodecyl and
the like. The phrase also includes branched chain isomers of straight chain
alkyl
groups, including but not limited to, the following which are provided by way
of
example: -CH(CH3)2, -CH(CH3)(CH2CH3), -CH(CH2CH3)2, -C(CH3)3, -C(CH2CH3)3,
-CH2CH(CH3)2, -CH2CH(CH3)(CH2CH3), -CH2CH(CH2CH3)2, -CH2C(CH3)3, -
CH2C(CH2CH3)3, -CH(CH3)CH(CH3)(CH2CH3), -CH2CH2CH(CH3)2, -
CH2CH2CH(CH3)(CH2CH3), -CH2CH2CH(CH2CH3)2, -CH2CH2C(CH3)3,
CH2CH2C(CH2CH3)3, -CH(CH3)CH2CH(CH3)2, -CH(CH3)CH(CH3)CH(CH3)2,
53
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
-CH(CH2CH3)CH(CH3)CH(CH3)(CH2CH3), and others. The phrase also includes
cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, and cyclooctyl and such rings substituted with straight and
branched
chain alkyl groups as defined above. The phrase also includes polycyclic alkyl
groups
such as, but not limited to, adamantyl norbornyl, and bicyclo[2.2.2]octyl and
such
rings substituted with straight and branched chain alkyl groups as defined
above.
Thus, the phrase unsubstituted alkyl groups includes primary alkyl groups,
secondary
alkyl groups, and tertiary alkyl groups. Unsubstituted allcyl groups may be
bonded to
one or more carbon atom(s), oxygen atom(s), nitrogen atom(s), and/or sulfur
atom(s)
in the parent compound. Preferred unsubstituted alkyl groups include straight
and
branched chain alkyl groups and cyclic alkyl groups having 1 to 20 carbon
atoms.
More preferred such unsubstituted alkyl groups have from 1 to 10 carbon atoms
while
even more preferred such groups have from 1 to 5 carbon atoms. Most preferred
unsubstituted alkyl groups include straight and branched chain alkyl groups
having
from 1 to 3 carbon atoms and include methyl, ethyl, propyl, and -CH(CH3)2.
The phrase "substituted alkyl" refers to an unsubstituted alkyl group as
defined above in which one or more bonds to a carbon(s) or hydrogen(s) are
replaced
by a bond to non-hydrogen and non-carbon atoms such as, but not limited to, a
halogen atom in halides such as F, Cl, Br, and I; and oxygen atom in groups
such as
hydroxyl groups, alkoxy groups, aryloxy groups, and ester groups; a sulfur
atom in
groups such as thiol groups, alkyl and aryl sulfide groups, sulfone groups,
sulfonyl
groups, and sulfoxide groups; a nitrogen atom in groups such as amines,
amides,
alkylamines, dialkylamines, arylainines, alkylarylamines, diarylamines, N-
oxides,
imides, and enamines; a silicon atom in groups such as in trialkylsilyl
groups,
dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and
other
heteroatoms in various other groups. Substituted alkyl groups also include
groups in
which one or more bonds to a carbon(s) or hydrogen(s) atom is replaced by a
bond to
a heteroatom such as oxygen in carbonyl, carboxyl, and ester groups; nitrogen
in
groups such as imines, oximes, hydrazones, and nitriles. Preferred substituted
alkyl
groups include, among others, allcyl groups in which one or more bonds to a
carbon or
hydrogen atom is/are replaced by one or more bonds to fluorine atoms. One
example
54
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
of a substituted alkyl group is the trifluoromethyl group and other alkyl
groups that
contain the trifluoromethyl group. Other alkyl groups include those in which
one or
more bonds to a carbon or hydrogen atom is replaced by a bond to an oxygen
atom
such that the substituted alkyl group contains a hydroxyl, alkoxy, aryloxy
group, or
heterocyclyloxy group. Still other alkyl groups include alkyl groups that have
an
amine, alkylamine, dialkylamine, arylamine, (alkyl)(aryl)amine, diarylamine,
heterocyclylamine, (alkyl)(heterocyclyl)amine, (aryl)(heterocyclyl)amine, or
diheterocyclylamine group.
The phrase "unsubstituted arylalkyl" refers to unsubstituted alkyl groups as
defined above in which a hydrogen or carbon bond of the unsubstituted alkyl
group is
replaced with a bond to an aryl group as defined above. For example, methyl (-
CH3)
is an unsubstituted alkyl group. If a hydrogen atom of the methyl group is
replaced
by a bond to a phenyl group, such as if the carbon of the methyl were bonded
to a
carbon of benzene, then the compound is an unsubstituted arylalkyl group
(i.e., a
benzyl group). Thus the phrase includes, but is not limited to, groups such as
benzyl,
diphenylmethyl, and 1-phenylethyl (-CH(C6H5)(CH3)) among others.
The phrase "substituted arylalkyl" has the same meaning with respect to
unsubstituted arylalkyl groups that substituted aryl groups had with respect
to
unsubstituted aryl groups. However, a substituted arylalkyl group also
includes
groups in which a carbon or hydrogen bond of the alkyl part of the group is
replaced
by a bond to a non-carbon or a non-hydrogen atom. Examples of substituted
arylalkyl
groups include, but are not limited to, -CH2C(=O)(C6H5), and -CH2(2-
methylphenyl)
among others.
A "pharmaceutically acceptable salt" includes a salt with an inorganic base,
organic base, inorganic acid, organic acid, or basic or acidic amino acid. As
salts of
inorganic bases, the invention includes, for example, alkali metals such as
sodium or
potassium; alkaline earth metals such as calcium and magnesium or aluminum;
and
ammonia. As salts of organic bases, the invention includes, for example,
trimethylamine, triethylamine, pyridine, picoline, ethanolamine,
diethanolamine, and
triethanolamine. As salts of inorganic acids, the instant invention includes,
for
example, hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and
phosphoric
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
acid. As salts of organic acids, the instant invention includes, for example,
formic
acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, lactic
acid, tartaric
acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic
acid,
benzenesulfonic acid, and p-toluenesulfonic acid. As salts of basic amino
acids, the
instant invention includes, for example, arginine, lysine and ornithine.
Acidic amino
acids include, for example, aspartic acid and glutamic acid.
"Treating" within the context of the instant invention, means an alleviation
of
symptoms associated with a biological condition, disorder, or disease, or halt
of further
progression or worsening of those symptoms, or prevention or prophylaxis of
the disease
or disorder. For example, within the context of treating patients having
pulmonary
hypertension, successful treatment may include a reduction direct vasodilation
of
pulmonary and/or systemic arterial vascular beds and inhibition of platelet
aggregation.
The result of this vasodilation will generally reduce right and left
ventricular afterload
and increased cardiac output and stroke volume. Dose-related negative
inotropic and
lusitropic effects can also result. The outward manifestation of these
physical effects can
include a decrease in the symptoms of hypertension, such as shortness of
breath, and an
increase in exercise capacity.
The present invention, thus generally described, will be understood more
readily by reference to the following examples, which are provided by way of
illustration and are not intended to be limiting of the present invention.
EXAMPLES
EXAMPLE I
In this Example, the bioavailability of treprostinil in rats after dosing
orally,
intraduodenally, intracolonically and via the portal vein was compared to
determine
possible barriers to bioavailability. In addition to bioavailability, a number
of
pharmacokinetic parameters were determined.
56
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Animal Dosing
The bioavailability of treprostinil was evaluated in Sprague-Dawley, male
rats.
Fifteen surgically modified rats were purchased from Hilltop Lab Animals
(Scottdale,
PA). The animals were shipped from Hilltop to Absorption Systems' West Chester
University facility (West Chester, PA), where they were housed for at least
twenty-
four hours prior to being used in the study. The animals were fasted for
approximately 16 hours prior to dosing. The fifteen rats used in this study
were
divided into five groups (I, II, III, IV and V).
The weight of the animals and the dosing regimen are presented in Table 1.
Table 1
Group Rat # Weight Route of Study Dose Volume Dose
(g) Administration Day (mL/k) (mg/kg)
118 327 Intravenous 0 2 1
I 119 329 Intravenous 0 2 1
120 320 Intravenous 0 2 1
121 337 Intraportal Vein 0 2 1
II 122 319 Intraportal Vein 0 2 1
123 330 Intraportal Vein 0 2 1
124 329 Intraduodenal 0 2 1
III 125 331 Intraduodenal 0 2 1
126 324 Intraduodenal 0 2 1
127 339 Intracolonic 0 2 1
IV 128 333 Intracolonic 0 2 1
129 320 Intracolonic 0 2 1
130 293 Oral 0 2 1
V 131 323 Oral 0 2 1
132 332 Oral 0 2 1
Samples were withdrawn at the following time points.
IV and IPV: 0 (pre-dose) 2, 5, 15, 30, 60, 120, 240, 360, 480 minutes
ID, IC and Oral: 0 (pre-dose), 5, 15, 30, 60, 120, 240, 360, 480 minutes
Approximately 0.50 to 0.75 mL of whole blood was collected from the jugular
vein of a cannulated rat. The blood was transferred to heparinized tubes and
placed
57
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
on ice until centrifuged. Following centrifugation the plasma was placed on
ice until
frozen at -70 ^ C prior to shipment to Absorption Systems
Analysis of plasma samples
Samples were analyzed using the following methodology:
Dosing Solution Preparation
The dosing solution was prepared by combining 15.2 mg of treprostinil
diethanolamine (12.0 mg of the free acid form) with 24 mL of 5% dextrose. The
solution was then sonicated until dissolved for a final concentration of 0.5
mg/mL.
The final pH of the dosing solution was 4.6. At the time of dosing, the dosing
solution was clear and homogenous.
Standards and Sample Preparation
To determine the concentration of treprostinil in rat plasma samples,
standards
were prepared with rat plasma collected in heparin obtained from Lampire
Biological
Laboratories (Lot #021335263) to contain 1000, 300, 100, 30, 10, 3, 1 and 0.3
ng/mL
of treprostinil. Plasma standards were treated identically to the plasma
samples.
Plasma samples were prepared by solid phase extraction. After an extraction
plate was equilibrated, 150 L of a plasma sample was placed into the well and
vacuumed through. The extraction bed was then washed with 600 L of
acetonitrile:
deionized water (25:75) with 0.2 % formic acid. The compound was eluted with
600
L of 90% acetonitrile and 10% ammonium acetate. The eluates were collected and
evaporated to dryness. The residue was reconstituted with 150 L of
acetonitrile:
deionized water (50:50) with 0.5 g/ml, of tolbutamide (used as an internal
standard).
58
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
HPLC Conditions
Column: Keystone Hypersil BDS C18 30 x 2 min i.d., 3 m.
Mobile Phase Buffer: 25 mM NH40H to pH 3.5 w/ 85% formic acid.
Reservoir A: 10% buffer and 90% water.
Reservoir B: 10% buffer and 90% acetonitrile.
Mobile Phase Composition:
Gradient Program:
Time Duration Grad. Curve % A % B
-0.1 0.10 0 80 20
0 3.00 1.0 10 90
3.00 1.00 1.0 0 100
4.00 2.00 0 80 20
Flow Rate: 300 L/min.
Inj. Vol.: 10 L
Run Time: 6.0 min.
Retention Time: 2.6 min.
Mass Spectrometer
Instrument: PE SCIEX API 2000
Interface: Electrospray ("Turbo Ion
Spray")
Mode: Multiple Reaction Monitoring
(MRM)
Precursor Ion. Product Ion
Treprostinil 389.2 331.2
IS 269.0 170.0
59
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Nebulizing Gas: 25 Drying Gas: 60, 350 C Curtain Gas: 25 Ion Spray:
-5000V
Orifice: -80 V Ring: -350V QO: IOV IQI: IIV
ST: 15V
R01: 11V IQ2: 35V R02: 40V IQ3: 55V R03: 45V
CAD Gas: 4
Method Validation
Table 2 lists the average recoveries (n=6) and coefficient of variation (c.v.)
for
rat plasma spiked with treprostinil. All samples were compared to a standard
curve
prepared in 50:50 dHZO:acetonitrile with 0.5 g/mL of tolbutamide to determine
the
percent of treprostinil recovered from the plasma.
Table 2: Accuracy and Precision of Method
Spiked Coefficient of
Concentration Percent Recovered Variation
1000 ng/mL 85.6 5.2
100 ng/mL 89.6 11.6
ng/mL 98.8 7.0
Pharmacokinetic Analysis
Pharmacokinetic analysis was performed on the average plasma concentration
for each time point.
The data were subjected to non-compartmental analysis using the
pharmacokinetic program WinNonlin v. 3.1 (2).
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
RESULTS
Clinical Observations
Prior to beginning the experiments it was noted that supra-pharmacological
doses of treprostinil would be needed to achieve plasma concentrations that
could be
analyzed with adequate sensitivity. Using the dose of 1 mg/kg some adverse
effects
were noted in animals dosed intravenously and via the intraportal vein.
All rats dosed intravenously displayed signs of extreme lethargy five minutes
after dosing but fully recovered to normal activity thirty minutes post-
dosing. In
addition, fifteen minutes after dosing all three animals dosed via the portal
vein
exhibited signs of lethargy. One rat (#123) expired before the thirty-minute
sample
was drawn. The other rats fully recovered. The remaining animals did not
display
any adverse reactions after administration of the compound.
Sample Analysis
Average plasma concentrations for each route of administration are shown in
Table 3.
Table 3
Average (n=3) plasma concentrations (ng/mL)
Time (min) Pre- 2 5 15 30 60 120 240 360 480
dose
Intravenous 0 1047.96 364.28 130.91 55.56 14.45 4.45 1.09 0.50 0.30
Intrap ortal Vein* 0 302.28 97.39 47.98 21.94 11.06 3.87 2.51 4.95 5.14
Intraduodenal 0 ---- 61.76 31.67 18.57 13.55 5.91 1.11 0.89 0.90
Intracolonic 0 ---- 7.46 3.43 3.52 1.48 0.64 0.36 0.06' 0.20'`
Oral 0 ---- 4.52 2.90 3.67 2.06 4.52 1.82 0.90 0.96
*n =2,
concentration falls below the limit of quantitation (LOQ) of the analytical
method
The plasma concentration versus time curves for intravenous, intraportal,
intraduodenal, intracolonic and oral dosing are shown in Figures 1 and 2.
Figure 3
shows the average plasma concentration versus time curves for all five routes
of
61
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
administration. In the experiments shown in these figures, the diethanolamine
salt
was used. Table 4 shows the pharmacokinetic parameters determined for
treprostinil.
The individual bioavailabilities of each rat are found in Table 5.
Table 4
Average Bioavailability and Pharmacokinetic Parameters of
Treprostinil in Rats
Average Average Volume of CLs
Route of 480 ~n C~a. TAX TU2 Bioavailability Distribution* (irL.miri'
AUC
Administration (min.nglrnL) (ngl"'L) (nvn) (min) (%) f SD (L.kg"') kg-')*
Intravenous 11253.49 2120'p 0 94 NA 1.98 88.54
Intraportal Vein 4531.74 302 2 ND 40.3 5.5 ND ND
Intraduodenal 2712.55 62 5 ND 24.1 '0.5 ND ND
Intracolonic 364.63 8 5 ND 3.2 2.5 ND ND
Oral 1036.23 5 5 ND 9.2 1.4 ND ND
*Normalized to the average weight of the rats
ND: Not determined
'PExtrapolated Value
62
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Table 5
Individual Bioavailabilities of Treprostinil in Rats
Route of Rat # Individual AUC480 min Individual Bioavailability
Administration (min.ng/mL) (%)
118 10302.85 NA
Intravenous 119 9981.52 NA
120 13510.65 NA
121 4970.67 44.2
Intraportal Vein 122 4093.21 36.4
123 ND ND
124 2725.68 24.2
Intraduodenal 125 2763.60 24.6
126 2646.05 23.5
127 72.63 0.7
Intracolonic 128 395.08 3.5
129 625.20 5.6
130 998.70 8.9
Oral 131 907.60 8.1
132 1203.73 10.7
NA: Not applicable
ND: Not determined
CONCLUSIONS
Treprostinil has a terminal plasma half-life of 94 minutes. The distribution
phase of treprostinil has a half-life of 10.3 minutes and over 90% of the
distribution
and elimination of the compound occurs by 60 minutes post-dosing. The volume
of
distribution (Vd = 1.98 L/kg) is greater than the total body water of the rat
(0.67 L/kg)
indicating extensive partitioning into tissues. The systemic clearance of
treprostinil
(88.54 mL/min/kg) is greater than the hepatic blood flow signifying that extra-
hepatic
clearance mechanisms are involved in the elimination of the compound.
63
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
First pass hepatic elimination of treprostinil results in an average
intraportal
vein bioavailability of 40.3%. Fast but incomplete absorption is observed
after
intraduodenal, intracolonic and oral dosing (Tmax < 5 min). By comparing the
intraportal vein (40.3%) and intraduodenal bioavailability (24.1%) it appears
that
approximately 60% of the compound is absorbed in the intestine. The average
intraduodenal bioavailibility is almost three times greater than the oral
bioavailibility
suggesting that degradation of treprostinil in the stomach or gastric emptying
may
influence the extent of systemic absorption.
Example 2
In this Example, Treprostinil concentrations were determined in male
Sprague-Dawley rats following a single oral dose of the following compounds:
O 0,CH2O6H5
O
HOI
OH
treprostinil benzyl ester
O 01H
O
0
~
H2N,`~01~,-T NH2
O
treprostinil diglycine
64
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
O O.CH
3
O
HOIi
-OH
treprostinil methyl ester
EXPERIMENTAL
Dosing Solution Preparation
All dosing vehicles were prepared less than 2 hours prior to dosing.
1. Treprostinil methyl ester
A solution of treprostinil methyl ester was prepared by dissolving 2.21 mg of
treprostinil methyl ester with 0.85 mL of dimethylacetamide (DMA). This
solution
was then diluted with 7.65 mL of PEG 400:Polysorbate 80:Water, 40:1:49. The
final
concentration of the dosing vehicle was 0.26 mg/mL of treprostinil methyl
ester
equivalent to 0.25 mg/mL of Treprostinil. The dosing vehicle was a clear
solution at
the time of dosing.
2. Treprostinil benzyl ester
A solution of treprostinil benzyl ester was prepared by dissolving 2.58 mg of
treprostinil benzyl ester with 0.84 mL of dimethylacetamide (DMA). This
solution
was then diluted with 7.54 mL of PEG 400:Polysorbate 80:Water, 40:1:49. The
final
concentration of the dosing vehicle was 0.268 mg/ml, of treprostinil benzyl
ester
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
equivalent to 0.25 mg/mL of Treprostinil. The dosing vehicle was a clear
solution at
the time of dosing.
3. Treprostinil diglycine
A solution of treprostinil diglycine was prepared by dissolving 1.86 mg of
compound with 0.58 mL of dimethylacetamide (DMA). This solution was then
diluted with 5.18 mL of PEG 400:Polysorbate 80:Water, 40:1:49. The final
concentration of the dosing vehicle was 0.323 mg/mL of treprostinil diglycine
equivalent to 0.25 mg/mL of Treprostinil. The dosing vehicle was a clear
solution at
the time of dosing.
Animal Dosing
The plasma concentrations of Treprostinil following administration of each
prodrug were evaluated in male Sprague-Dawley rats. Rats were purchased from
Hilltop Lab Animals (Scottdale, PA). The animals were shipped from Hilltop to
Absorption Systems' West Chester University facility (West Chester, PA). They
were housed for at least twenty-four hours prior to being used in the study.
The
animals were fasted for approximately 16 hours prior to dosing. The rats used
in this
study were divided into three groups (I, II and III). Groups I - III were
dosed on the
same day.
The weight of the animals and the dosing regimen are presented in Table 6.
66
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Table 6 - Study Design
Weight, Route of Compound Dose Volume Dose*
Group Rat#
(kg) Administration Dosed (mL/k) (mg/kg)
638 306 Oral
I 639 310 Oral Treprostinil 2 0.520
640 319 Oral methyl ester
641 319 Oral
II 642 309 Oral Treprostinil 2 0.616
643 320 Oral benzyl ester
644 318 Oral Treprostinil
III 645 313 Oral diglycine 2 0.646
646 322 Oral
* This dose ofprodrug = 0.500 nzg/kg of the active, Treprostinil
Animals were dosed via oral gavage. Blood samples were taken from a
jugular vein cannula at the following time points:
0 (pre-dose) 5, 15, 30, 60, 120, 240, 360 and 480 minutes
The blood samples were withdrawn and placed into tubes containing 30 L of
a solution of 500 units per mL of heparin in saline, and centrifuged at 13,000
rpm for
minutes. Approximately 200 L of plasma was then removed and dispensed into
appropriately labeled polypropylene tubes containing 4 L of acetic acid in
order to
stabilize any prodrug remaining in the samples. The plasma samples were frozen
at -
C and were transported on ice to Absorption Systems Exton Facility. There they
were stored in a -80 C freezer pending analysis.
Analysis of plasma samples
Plasma samples were analyzed as described in Example 1. In brief,
Treprostinil was extracted from the plasma via liquid-liquid extraction then
analyzed
by LC/MS/MS. The analytical validation results were reported previously in
Example
1. The lower limit of quantification (LLOQ) of the analytical method was 0.01
ng/mL. Samples were not assayed for unchanged prodrug.
67
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Acceptance Criteria for Analytical Runs
Two standard curves, with a minimum of five points per curve, and a
minimum of two quality control samples (QCs) were dispersed throughout each
run.
Each route of administration was bracketed by a standard curve used for back-
calculation. The standards and QCs must be within :L 15% (20% for the LLOQ)
accuracy and precision for the run to be accepted. At least 75% of all
standards and
QCs must pass the acceptance criteria.
Pharmacokinetic Analysis
Pharmacokinetic analysis was performed on the plasma concentration of
Treprostinil for each individual rat at each time point and on the average
plasma
concentration for all three rats in the group for each time point. The data
were
subjected to non-compartmental analysis using the pharmacokinetic program
WinNonLin v. 3.1 (2).
RESULTS
Study Observations
No adverse reactions were observed following oral administration of
treprostinil methyl ester, treprostinil benzyl ester or treprostinil
diglycine.
Plasma Stability of prodrugs in Acidified Rat Plasma
In order to terminate any conversion of prodrug to active after samples were
withdrawn the plasma was acidified. Acetic acid (v/v) was added to each plasma
sample immediately after centrifugation of the red blood cells to a
concentration of
2%. In-vitro plasma stability of each prodrug was performed to insure that the
compound was stable in acidified plasma. To perform this assay 2% acetic acid
was
added to blank rat plasma obtained from Lampire Biological. The acidified rat
68
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
plasma was equilibrated at 37 C for three minutes prior to addition of
prodrug. The
initial concentration of each prodrug was 1000 ng/mL. A 100 L aliquot of
plasma
(n=3 per time point) was taken at 0, 60 and 120 minutes. Each aliquot was
combined
with 20 L of HCl and vortexed. Liquid-liquid extraction was then performed
and the
concentration of Treprostinil in each sample determined. The concentration of
Treprostinil at each time point in acidified rat plasma is given in Table 7.
Small
amounts of Treprostinil appear to be present in the neat compound sample of
treprostinil methyl ester and treprostinil diglycine. The concentration of
Treprostinil
remained constant throughout the course of the experiment, indicating that
there was
no conversion of prodrug into active compound occurring in acidified plasma.
Table 7 - Plasma Stability of Prodrugs in Acidified Dog Plasma
Treprostinil Concentration (ng/mL) SD (n=3)
Time (min) Treprostinil Treprostinil Treprostinil
methyl ester benzyl ester diglycine
0 56.8 9.3 <0.01 54.9 4.3
60 55.1 5.0 <0.01 51.8 5.9
120 53.8 1.3 <0.01 54.5 0.8
Total %Treprostinil 5.7 < 0.01 5.5
Average Treprostinil plasma concentrations following administration of
treprostinil methyl ester, treprostinil benzyl ester or treprostinil diglycine
are shown in
Table 8.
69
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Table 8 - Treprostinil Concentrations (Average SD (n=3) Plasma
Concentrations (ng/mL)
Oral Dosing Pre- 5 15 30 60 120 240 360 480
Solution Dose (min) (min) (min) (min) (min) (min) (min) (min)
Treprostinil 0 < 0.01 0.2 0.3 0.5 1.5 0.2 0.1
methyl ester 0.0 0.1 0.1 0.8 0.7 < 0.01 0.1
Treprostinil 0 3.1 1.9 2.5 3.2 7.3 1.6 0.4 0.6
benzyl ester 2.8 0.8 1.5 1.9 4.9 1.2 0.40 0.9
Treprostinil 0 < 0.01 1.1 6.6 0.5 40. 9.0 2.1 1.3
diglycine 1.9 10.7 0.3* 5.8 13.5 2.9 0.8
* Due to insufficient amount of sample collected this time point is the
average of n=2
rats.
Figures 4-7 contain graphical representations of the plasma concentration
versus time curves for Treprostinil in rat following administration of each
prodrug.
Table 9 lists each figure and the information displayed.
Table 9 - List of Figures
Figure Description
4 Oral Dose of Treprostinil methyl ester
Oral Dose of Treprostinil'benzyl ester
6 Oral Dose of Treprostinil diglycine
7 Oral Dose of Treprostinil benzyl ester and
Treprostinil diglycine Compared to
Treprostinil Alone from Example 1
Pharmacokinetic Analysis
Bioavailability of the prodrug was determined relative to that of the active
compound based on Example 1 in which Treprostinil was dosed to rats. The
following formula was used to determine relative bioavailability (F):
Relative F = (AUC(prodrug Dose)/Dose)I(AUC(Treprostinil Dose)/DDSe)* 100
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Bioavailability was also determined relative to an intravenous dose of
Treprostinil in rats determined in Example 1. Results are listed in Table 10.
Table 10 - Average Relative Bioavailability and Pharmacokinetic
Parameters of Treprostinil in Rats
Test Dose, Average Cmax Tmax Relative Bioavailability
Compound AUC .t Bioavailability o
Administered (mg/kg) (m n.ng/mL) (ng/mL) (min) (%) SD (n=3) (~ ) SD (n=3)
Treprostinil 0.5 212 1.50 120 41.0 16 3.8 2
methyl ester
Treprostinil 0.5 1171 7.20 120 226 155 20.8 14
benzyl ester
Treprostinil 0.5 2242 9.04 240 433 631 39.9 58
diglycine
CONCLUSIONS
In this study the relative oral bioavailabilities of prodrugs of Treprostinil
were
determined in rats. Treprostinil methyl ester resulted in Treprostinil area
under the
plasma concentration versus time curves (AUCs) less than that after dosing the
active
compound. Prodrugs treprostinil benzyl ester and treprostinil diglycine both
had
Treprostinil average AUCs greater than that after dosing of the active
compound.
Treprostinil diglycine had the highest relative bioavailability of 433% with
over 4
times more Treprostinil reaching the systemic circulation. The Cmax of 9 ng/mL
of
Treprostinil following administration of treprostinil diglycine occurred at
240 minutes
post-dosing. The Cmax following dosing of Treprostinil is 5 ng/mL and occurs
only
minutes post-dosing. Treprostinil benzyl ester had a relative bioavailability
of 226
155 % with a Cmax of 7.2 ng/mL occurring 120 minutes post-dosing. It should
also
be noted that the AUCs are not extrapolated to infinity.
71
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
REFERENCES
1. WinNonlin User's Guide, version 3.1, 1998-1999, Pharsight Co., Mountain
View, CA 94040.
Example 3
This example illustrates a pharmacokinetic study of treprostinil following
administration of a single duodenal dose of treprostinil and various prodrugs
of the
present invention.
In this study, the area under the curve of Treprostinil in male Sprague-Dawley
rats following a single intraduodenal dose of treprostinil monophosphate
(ring),
treprostinil monovaline (ring), treprostinil monoalanine (ring) or
treprostinil
monoalanine (chain), prodrugs of treprostinil was compared. The compounds were
as
follows:
H
OR3
-11111OR2
H
OCH2CO2R1
having the following substituents:
Compound R R R
treprostinil monophosphate H -P03H3 H
(ring)
treprostinil monovaline H -COCH(CH(CH3)2)NH2 H
(ring)
treprostinil monoalanine H -COCH(CH3)NH2 H
(ring)
treprostinil monoalanine H H -COCH(CH3)NH2
(chain)
72
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
EXPERIMENTAL
Dosing Solution Preparation
All dosing vehicles were prepared less than 2 hours prior to dosing.
1. treprostinil monophosphate (ring)
A dosing solution of treprostinil monophosphate (ring) was prepared by
dissolving 1.01 mg of treprostinil monophosphate (ring) in 0.167 mL of
dimethylacetamide (DMA) until dissolved. This solution was further diluted
with 1.50
mL of PEG 400: Polysorbate 80: Water, 40: 1: 49. The final concentration of
the
dosing vehicle was 0.603 ing/mL of prodrug equivalent to 0.5 mg/mL of
Treprostinil.
The dosing vehicle was a clear solution at the time of dosing.
2. treprostinil monovaline (ring)
A 50 mg/mL solution of treprostinil monovaline (ring) was prepared in
dimethylacetamide (DMA). A 25 L aliquot of the 50 mg/mL stock solution was
then
diluted with 175 p,L of DMA and 1.8 mL of PEG 400: Polysorbate 80: Water, 40:
1:
49. The final concentration of the dosing vehicle was 0.625 mg/mL of prodrug
equivalent to 0.5 mg/mL of Treprostinil. The dosing vehicle was a clear
solution at
the time of dosing.
3. treprostinil monoalanine (ring)
A solution of treprostinil monoalanine (ring) was prepared by dissolving 1.05
mg of treprostinil monoalanine (ring) in 0.178 mL of dimethylacetamide (DMA)
until
dissolved. This solution was further diluted with 1.60 mL of PEG 400:
Polysorbate
80: Water, 40: 1: 49. The final concentration of the dosing vehicle was 0.590
mg/mL
73
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
of treprostinil monoalanine (ring) equivalent to 0.5 mg/mL of Treprostinil.
The dosing
vehicle was a clear solution at the time of dosing.
4. treprostinil monoalanine (chain)
A solution of treprostinil monoalanine (chain) was prepared by dissolving 0.83
mg of treprostinil monoalanine (chain) in 0.14 mL of dimethylacetamide (DMA)
until
dissolved. This solution was further diluted with 1.26 mL of PEG 400:
Polysorbate
80: Water, 40: 1: 49. The final concentration of the dosing vehicle was 0.591
mg/mL
of treprostinil monoalanine (chain) equivalent to 0.5 mg/mL of Treprostinil.
The
dosing vehicle was a clear solution at the time of dosing.
Animal Dosing
The plasma concentrations of Treprostinil following oral administration of
each prodrug were evaluated in male Sprague-Dawley rats. Twelve rats were
purchased from Hilltop Lab Animals (Scottdale, PA). The animals were shipped
from
Hilltop to Absorption Systems' West Chester University facility (West Chester,
PA).
They were housed for at least twenty-four hours prior to being used in the
study. The
animals were fasted for approximately 16 hours prior to dosing. The twelve
rats used
in this study were divided into four groups. All groups were dosed on day 1 of
the
study. The weight of the animals and the dosing regimen are presented in Table
11.
74
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
TABLE 11
Rat # Weight (g) Compound Dose Volume Dose*
(mL/kg) (mg/kg)
130 327 treprostinil mono phosphate (ring) 1 0.603
131 321 treprostinil monophosphate (ring) 1 0.603
132 310 treprostinil monophosphate (ring) 1 0.603
133 328 tre rostinil monovaline (ring) 1 0.625
134 326 treprostinil monovaline (ring) 1 0.625
135 346 treprostinil monovaline (ring) 1 0.625
136 321 treprostinil monoalanine (chain) 1 0.591
137 319 treprostinil monoalanine (chain) 1 0.591
138 330 treprostinil monoalanine (chain) 1 0.591
139 316 treprostinil monoalanine (ring) 1 0.590
140 330 treprostinil monoalanine (ring) 1 0.590
141 339 treprostinil monoalanine (ring) 1 0.590
* This dose ofprodrug = 0.500 ing/kg of treprostinil
Animals were dosed via an indwelling duodenal cannula. Blood samples were
takenfrom a jugular vein cannula at the following time points:0 (pre-dose) 5,
15, 30,
60, 120, 240, 360 and 480 minutes.
The blood samples were withdrawn and placed into tubes containing 30 L of
a solution of 500 units per mL of heparin in saline, and centrifuged at 13,000
rpm for
minutes. Approximately 200 L of plasma was then removed and dispensed into
appropriately labeled polypropylene tubes containing 4 L of acetic acid in
order to
stabilize any prodrug remaining in the samples. The plasma samples were frozen
at -
C and were transported on ice to Absorption Systems Exton Facility. There they
were stored in a -80 C freezer pending analysis.
Analysis of plasma samples
Plasma samples were analyzed using the methods described above. In brief,
Treprostinil was extracted from the plasma via solid phase extraction then
analyzed
by LC/MS/MS. The lower limit of quantification (LLOQ) of the analytical method
was 0.03 ng/mL.
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Acceptance Criteria for Analytical Runs
Four standard curves, with a minimum of five points per curve, and a
minimum of two quality control samples (QCs) at 3 concentrations were
dispersed
throughout each run. Each prodrug set was bracketed by a standard curve used
for
back-calculation. The standards and QCs must be within d 15% (20% for the
LLOQ)
accuracy and precision for the run to be accepted. At least 75% of all
standards and
QCs must pass the acceptance criteria.
Pharmacokinetic Analysis
Pharmacokinetic analysis was performed on the plasma concentration of
Treprostinil for each individual rat at each time point and on the average
plasma
concentration for all three rats in the group for each time point.
The data were subjected to non-compartmental analysis using the
pharmacokinetic program WinNonLin v. 3.1 (2).
RESULTS
Study Observations
No adverse reactions were observed following intraduodenal administration of
treprostinil monophosphate (ring), treprostinil monovaline (ring),
treprostinil
monoalanine (ring) or treprostinil monoalanine (chain).
Ex-Vivo Plasma Stability of prodrugs in Acidified Rat Plasma
In order to terminate any conversion of prodrug to active after samples were
withdrawn, the plasma was acidified. Acetic acid (v/v) was added to each
plasma
sample immediately after separation of the red blood cells to a concentration
of 2%.
In-vitro plasma stability of each prodrug was performed to insure that the
compound
76
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
was stable in acidified plasma. To perform this assay 2% acetic acid was added
to
blank rat plasma obtained from Lampire Biological. The acidified rat plasma
was
brought to room temperature for three minutes prior to addition of prodrug.
The initial
concentration of each prodrug was 1000 ng/mL. A 100 L aliquot of plasma (n=3
per
time point) was taken at 0, 60 and 120 minutes. Sample preparation of each
plasma
sample was performed as described above and the concentration of Treprostinil
monitored.
Treprostinil concentrations did not increase in any of the acidified plasma
samples spiked with prodrug over the two-hour period of the experiment.
Sample Analysis
Average Treprostinil plasma concentrations following administration of
treprostinil monophosphate (ring), treprostinil monovaline (ring),
treprostinil
monoalanine (ring) or treprostinil monoalanine (chain) are shown in Table 12.
TABLE 12: AVERAGE SD (N=3)
PLASMA TREPROSTINIL CONCENTRATIONS (NG/ML)
Oral Dosing Pre- 5 15 30 60 120 240 360 480
Solution dose (min) (miii) (min) (min) (min) (min) (min) (min)
treprostinil 0 8.62 6.57 3.31 4.31 2.07 0.91 0.26 0.3
monophosphate 3.0 1.7 1.2 0.8 0.4 0.5 0.08 0.08
(ring)
treprostinil 0 0.76 0.91 1.52 1.53 1.65 0.66 0.15 ' 0.05
monovaline (ring) 0.2 0.7 0.6 0.6 0.7 0.1 0.03 0.02
treprostinil 0 2.42 2.52 2.91 3.25 1.69 0.55 0.20 0.22
monoalanine 0.6 0.4 0.6 1.5 0.4 0.2 0.1 0.2
(ring)
treprostinil 0 9.53 3.92 3.83 2.74 0.86 0.29 0.08 0.19
monoalanine 2.6 0.6 0.7 0.9 0.4 0.2 0.04 0.3
(chain)
77
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Figures 8-12 contain graphical representations of the plasma concentration
versus time curves for Treprostinil in rat following administration of each
prodrug.
Table 13 lists each figure and the information displayed.
TABLE 13
Figure Description
8 Intraduodenal dose of treprostinil mono hos hate (ring)
9 Intraduodenal dose of treprostinil monovaline (ring)
Intraduodenal dose of treprostinil monoalanine (ring)
11 Intraduodenal dose of treprostinil monoalanine (chain)
12 Intraduodenal dose of each prodrugcompared to treprostinil alone from
Example 1
Pharmacokinetic Analysis
Bioavailability of the prodrug was determined relative to that of the active
compound based on a previous study in which Treprostinil was dosed to rats.
The
following formula was used to determine relative bioavailability (F):
Relative F = (AUC(prodrugDose)/Dose)/(AUC(Treprostinil Dose)/DDse)* 100
Absolute bioavailability was also estimated using data from an intravenous
dose of Treprostinil in rats determined in Example 1. Results are listed in
Table 14.
Table 14
List of Figures
Figure Description
8 Intraduodenal Dose of treprostinil monophosphate (ring)
9 Intraduodenal Dose of treprostinil monovaline (ring)
10 Intraduodenal Dose of treprostinil monoalanine (ring)
11 Intraduodenal Dose of treprostinil monoalanine (chain)
12 Intraduodenal Dose of Each Prodrug Compared to
Treprostinil Alone from Example 1
78
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
CONCLUSIONS
The relative intraduodenal bioavailabilities of four prodrugs of Treprostinil
were determined in rats. All the compounds had relative intraduodenal
bioavailabilities less than that of the active compound. treprostinil
monophosphate
(ring) and treprostinil monoalanine (ring) had the highest relative
intraduodenal
bioavailability at 56% and 38% respectively. The Tmax for treprostinil
monophosphate (ring) and treprostinil monoalanine (chain) occurred 5 minutes
post-
dosing. treprostinil monovaline (ring) and treprostinil monoalanine (ring) had
longer
absorption times with Tmax values of 120 and 60 minutes respectively. Maximum
Treprostinil concentrations were highest following treprostinil monophosphate
(ring)
and treprostinil monoalanine (chain) dosing. They reached approximately 9
ng/mL 5
minutes post-dosing. The bioavailabilities are much greater when dosed
intraduodenally than when dosed orally as measured by treprostinil plasma
levels.
REFERENCES
1. WinNonlin User's Guide, version 3.1, 1998-1999, Pharsight Co., Mountain
View, CA 94040.
Example 4
In this Example, Treprostinil concentrations will be determined in male
Sprague-Dawley rats following a single oral or intraduodenal dose of the
following
compounds of structure II:
H
OR3
H
OCH2CO2R1
79
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
having the following substituents:
Cpd. R' R : R.
A -CH2CONH2 H H
B -CH2CON(CH2)20H H H
C -CH2CON(CH3)2 H H
D -CH2CONHOH H H
E -CH2C6H4NO2 (p)* H H
F -CH2C6H4OCH3 (p)* H H
G -CH2C6H4C1(o)* H H
H CH2C6H4 02)2 (0,P)* H H
I -CH2C6H4F ( )* H H
J H -P03H3 H
K H H -P03H3
L H -COCH2NH2 H
M H H -COCH2NH2
N H -COCH(CH3)NH2 H
H H -COCH(CH3)NH2
P H -COCH(CH3)NH2 -COCH(CH3)NH2
* - o denotes ortho substitution, in denotes meta substitution and p denotes
para
substitution.
Examples of these compounds include:
NHZ Q"~( 0.\\// X ,-,,,_,,,OH
Q~~( a I0N
0 0
OH
HOum
.......
;""".OH
"'OH c H37NO6 Exact aass:15535.31
Exact Mass: 447.26
Mol. Wt.: 447.56 C, 65.02; H b 8 Mo.447; 7; N, 2.61; 5C, 67.09; H, 8.33; N,
3.13; 0, 21.45 0, 23.89
Treprostinil glycolamide ester TreprostinilN,N-diethanol glycolamide ester
A B
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
0 I0I
n J~ 0 l~ OH
H
O 0
H01,11". H0n11 ,
OH OH
C27H4IN06 CuH37NO7
Exact Mass: 475.29 Exact Mass: 463.26
Mol. Wt.: 475.62 Mol. Wt.: 463.56
t 68.18;g8.69;N,2.94;0,20.18 ty64.77;H 8.05; N, 3.02; 0, 24.16
Treprostinil NN-dimthyl glycolamide Treprostinil N-hydroxy glycolamide ester
D
0 NH2
.,,uut10
H 0 H2 Y
=-III11O
H
O
O OH
C29H44N207
Exact Mass: 532.31
Mol. Wt.: 532.67
C, 65.39; H, 8.33; N, 5.26; 0, 21.03
P
Prodrug preparation and analysis will take place as described in Examples 1
and 2 above. Additionally, the oral bioavailability of treprostinil,
treprostinil sodium
81
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
and the compounds shown in Example 2 and this Example will be administered in
close proximity to or simultaneously with various different p-glycoprotein
inhibiting
compounds at varying concentrations and tested to determine the effect of the
p-
glycoprotein inhibitors on the oral bioavailability of the compounds. The p-
glycoprotein inhibitors will be administered both intravenously and orally.
Example 5
Clinical Studies with Treprostinil Diethanolamine
Introduction
Prior to proceeding directly into clinical studies with a sustained release
(SR)
solid dosage form of UT-15C (treprostinil diethanolamine), a determination of
the
pharmacokinetics of an oral "immediate release" solution was performed. The
first
clinical study (01-101) evaluated the ability of escalating doses of an oral
solution of
UT-15C to reach detectable levels in plasma, potential dose-plasma
concentration
relationship, bioavailability and the overall safety of UT-15C. Volunteers
were dosed
with the solutions in a manner that simulated a sustained release formulation
releasing
drug over approximately 8 hours.
The second clinical study (01-102) assessed the ability of two SR solid dosage
form prototypes (i.e., 1. microparticulate beads in a capsule and, 2. tablet)
to reach
detectable levels in plasma and the potential influence of food on these
plasma drug
concentrations. The SR prototypes were designed to release UT-15C over
approximately an 8 hour time period.
Details of the two clinical studies are described below.
82
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Clinical Study 01-101
A Safety, Tolerability, and Pharinacokinetic Study of Multiple Escalating
Doses of UT-15C (Treprostinil Diethanolamine) Administered as an Oral Solution
in
Healthy Adult Volunteers (Including Study of Bioavailability).
The oral solution of UT-15C was administered to 24 healthy volunteers to
assess the safety and pharmacokinetic profile of UT-15C as well as its
bioavailability.
To mimic a SR release profile, doses were administered every two hours for
four
doses at either 0.05 mg per dose (total = 0.2 mg), 0.125 mg per dose (total =
0.5 mg),
0.25 mg per dose (total = 1.0 mg), or 0.5 mg per dose (total = 2.0 mg). Study
endpoints included standard safety assessments (adverse events, vital signs,
laboratory
parameters, physical examinations, and electrocardiograms) as well as
pharmacokinetic parameters.
All subjects received all four scheduled doses and completed the study in its
entirety. Treprostinil plasma concentrations were detectable in all subjects
following
administration of an oral solution dose of UT-15C. Both AUC;,,f and Cmax
increased
in a linear fashion with dose for each of the four dose aliquots. The highest
concentration observed in this study was 5.5ing/mL after the third 0.25 mg
solution
dose aliquot of the 2.0 mg UT-15C total dose. Based on historical intravenous
treprostinil sodium data, the mean absolute bioavailability values for the 0.2
mg, 0.5
mg, 1.0 mg and 2.0 mg doses of UT-15C were estimated to be 21%, 23%, 24% and
25%, respectively. The results of this study are respectively shown in Figures
13A-
13D.
UT-15C was well tolerated by the majority of subjects at all doses given.
There were no clinically significant, treatment emergent changes in
hematology,
clinical chemistry, urinalysis, vital signs, physical exams, and ECGs. The
most
frequently reported adverse events were flushing, headache, and dizziness.
This
safety profile with UT-15C (treprostinil diethanolamine) is consistent with
the
reported safety profile and product labeling of Remodulin (treprostinil
sodium) and
other prostacyclin analogs. Thus, changing the salt form of treprostinil did
not result
83
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
in any unexpected safety issues following the protocol specified dosing
regimen (i.e.
single dose every 2 hours for four total doses on a single day).
Clinical Study 01-102
A Safety, Tolerability, and Fharmacokinetic Study Comparing a Single Dose
of a Sustained Release Capsule and Tablet Formulation of UT-15C (Treprostinil
Diethanolamine) Administered to Healthy Adult Volunteers in the Fasted and Fed
State
The 01-102 study was designed to evaluate and compare the safety and
pharmacokinetic profiles of a (1) UT-15C SR tablet prototype and, (2) UT-15C
SR
capsule prototype (microparticulate beads in a capsule) in both the fasted and
fed
state. Each of the SR dosage forms weres designed to release UT-15C (1 mg)
over an
approximate 8-hour time period. Fourteen healthy adult volunteers were
assigned to
receive the SR tablet formulation while an additional fourteen volunteers were
assigned to receive the SR capsule formulation. Subjects were randomized to
receive
a single dose (1 mg) of their assigned SR prototype in both the fasted and fed
state. A
crossover design was employed with a seven day wash-out period separating the
fed/fasted states. For the fed portion of the study, subjects received a high
calorie,
high fat meal. Study endpoints included standard safety assessments (adverse
events,
vital signs, laboratory parameters, physical examinations, and
electrocardiograms) as
well as pharmacokinetic parameters.
All subjects administered UT-15C SR tablets and capsules had detectable
treprostinil plasma concentrations. Calculations of area under the curve from
zero to
twenty-four hours (AUCO_24) indicate that total exposure to UT-15C SR occurred
in
the following order: Tablet Fed > Capsule Fasted > Tablet Fasted > Capsule
Fed.
Figure 14 displays the pharmacokinetic profiles of the two formulations in the
fasted
and fed states.
84
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
UT-15C SR tablets and capsules were tolerated by the majority of subjects.
All adverse events were mild to moderate in severity and were similar to those
described in Study 01-101 and in Remodulin's product labeling. Additionally,
there
were no treatment-emergent changes in vital signs, laboratory parameters,
physical
examinations, or electrocardiograms throughout the study.
These results demonstrate that detectable and potentially therapeutic drug
concentrations can be obtained from a solid dosage form of UT-15C and that
these
concentrations can be maintained over an extended period of time through
sustained
release formulation technology.
POLYMORPHS OF TREPROSTINIL DIETHANOLAMINE
Two crystalline forms of UT-15C were identified as well as an amorphous
form. The first, which is metastable, is termed Form A. The second, which is
thermodynamically more stable, is Form E. Each form was characterized and
interconversion studies were conducted to demonstrate which form was
thermodynamically stable. Form A is made according to the methods in Table 15.
Form B is made from Form A, in accordance with the procedures of Table 16.
Table 15
Solvent Conditionsa Habit/Description XRPD Sample
Resultb ID
tetrahydrofuran FE opaque white solids; morphology A 1440-
unknown, birefrin ent 72-02
SE glassy transparent solids A (PO) 1440-
72-03
SC (60 C) translucent, colorless glassy sheets A 1440-
of material, birefrin ent 72-16
Toluene slurry (RT), 6d white solids; opaque masses of A + B 1440-
smaller particles 72-01
toluene:IPA SC(60 C) white solids; spherical clusters of A 1480-
(11.4:1) fibers, birefrin ent 21-03
Water FE opaque white solids; morphology A 1440-
unknowm, birefrin ent 72-07
SE opaque ue ring of solids, birefringent A + B 1440-
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
72-08
freeze dry white, glassy transparent solids A+ B 1480-
58-02
water:ethanol FE opaque white solids; morphology A + 1440-
(1:1) unknown, birefringent 11.5 pk 72-09
FE clear and oily substance with some B 1480-
opaque solids 79-02
SE glassy opaque ring of solid A 1440-
72-10
a. FE = fast evaporation; SE = slow evaporation; SC = slow cool
b. IS = insufficient sample; PO = preferred orientation; LC = low
crystallinity; plc = peak
c. XRPD = X-ray powder diffraction
Table 16
Solvent Conditions Habit/Description XRPD Sample ID
Result
ethanol/ water FE glassy appearing solids of _b 1519-68-01
(1:1) unknown morphology;
birefringent
1,4-dioxane slurry(50 C), 6d white solids; opaque masses of B 1519-73-02 a
material; morphology unknown
slurry(50 C), 2d small grainy solids; with B 1557-12-01
birefringence
subsample of - B 1557-15-01
1557-12-01
subsample of white solids B 1557-15-02
1557-12-01
slu (50 C), 2d - B 1557-17-01
iso ro anol slurry(RT), Id white solids 1519-96-03
tetrahydrofuran sl RT , I d - - 1519-96-02
toluene slu 50 C), 6d white solids B 1519-73-01
a. Seeds of sample #1480-58-01 (A+B) added
b. Samples not analyzed
86
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Characterization of Crystal Forms:
Form A
The initial material synthesized (termed Form A) was characterized using X-
ray powder diffraction (XRPD), differential scanning calorimetry (DSC),
thermogravimetry (TG), hot stage microscopy, infrared (IR) and Raman
spectroscopy,
and moisture sorption. Representative XRPD of Form A is shown in Figure 15.
The
IR and Raman spectra for Form A are shown in Figures 16 and 17, respectively.
The
thermal data for Form A are shown in Figure 18. The DSC thernogram shows an
endotherm at 103 C that is consistent with melting (from hot stage
microscopy). The
sample was observed to recrystallize to needles on cooling from the melt. The
TG
data shows no measurable weight loss up to 100 C, indicating that the
material is not
solvated. The moisture sorption data are shown graphically in Figure 19. Form
A
material shows significant weight gain (>33%) during the course of the
experiment
(beginning between 65 to 75% RH), indicating that the material is hygroscopic.
In
addition, hygroscopicity of treprostinil diethanolamine was evaluated in
humidity
chambers at approximately 52% RH and 68% RH. The materials were observed to
gain 4.9% and 28% weight after 23 days in the -52% RH and - 68% RH chambers,
respectively.
Based on the above characterization data, Form A is a crystalline, anhydrous
material which is hygroscopic and melts at 103 C.
Form B
Treprostinil diethanolamine Form B was made from heated slurries (50 C) of
Form A in 1,4 dioxane and toluene, as shown in Table 16. Material isolated
from 1,4-
dioxane was used to fully characterize Form B. A representative XRPD pattern
of
Form B is shown in Figure 20. Form A and Form B XRPD patterns are similar,
87
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
however, significant differences are observed in the range of approximately 12
- 17
2 0 (Figure 20).
The thermal data for Form B are shown in Figure 21. The DSC thermogram
(Sample ID 1557-17-01) shows a single endotherm at 107 C that is consistent
with a
melting event (as determined by hotstage microscopy). The TG shows minimal
weight loss up to 100 C.
The moisture sorption/desorption data for Form B are shown in Figure 22.
There is minimal weight loss at 5% RH and the material absorbs approximately
49%
water at 95% RH. Upon desorption from 95% down to 5% RH, the sample loses
approximately 47%.
Form A and Form B can easily be detected in the DSC curve. Based on the
above characterization data, Form B appears to be a crystalline material which
melts
at 107 C.
88
CA 02526534 2005-11-21
WO 2005/007081 PCT/US2004/016401
Thermodynamic Properties:
Inter-conversion experiments were carried out in order to determine the
thermodynamically most stable form at various temperatures. These studies were
performed in two different solvents, using Forms A and B material, and the
data are
summarized in Table 17. Experiments in isopropanol exhibit full conversion to
Form
B at ambient, 15 C, and 30 C after 7 days, 11 days, and 1 day, respectively.
Experiments in tetrahydrofuran also exhibit conversion to Form B at ambient,
15 C,
and 30 C conditions. Full conversion was obtained after 11 days at 15 C, and
I day
at 30 C. At ambient conditions, however, a minor amount of Form A remained
after
7 days based on XRPD data obtained. Full conversion would likely occur upon
extended slurry time. Based on these slurry inter-conversion experiments, Form
B
appears to be the most thermodynamically stable form. Form A and Form B appear
to
be related monotropically with Form B being more thermodynamically stable.
Table 17
Interconversion Studies of Treprostinil Diethanolamine
Sample No. Forms Solvent Experiment/ Temperature Time
Starting Materials
1557-22- A vs. B isopropanol solid mixture ambient 7 days
01 # 1557-20-01 a
1557-47- A vs. B solid mixture 15 C 11 days
02 # 1557-35-01 d
1557-33- A vs. B solid mixture 30 C 1 day
02 # 1557-35-Old
1557-21- A vs. B solid mixture 50 C -
02e # 1557-20-0 lb
1557-20- A vs. B tetrahydrofuran solid mixture ambient 7 days
03 # 1557-20-01
1557-47- A vs. B solid mixture 15 C 11 days
01 # 1557-35-Old
1557-33- A vs. B solid mixture 30 C 1 day
01 # 1557-35-old
1557-21- A vs. B solid mixture 50 C -
Ole # 1557-20-0l
a. saturated solution Sample ID 1557-21-03
b. saturated solution Sample ID 1519-96-03
c. saturated solution Sample ID 1519-96-02
d. saturated solution prepared just prior to addition of solids
89
CA 02526534 2010-01-11
WO 2005/007081 PCT/US2004/0164111
e. samples not analyzed as solubility (at 50 C) of treprostinil
diethanolamine was very high and solutions became
discolored.
While preferred embodiments have been illustrated and described, it should be
understood that changes and modifications can be made therein in accordance
with
ordinary skill in the art without departing from the invention in its broader
aspects as
defined herein.
